Thermo Scientific Molecular Biology Solutions 2012 - 2013 - Biocenter

Thermo Scientific Molecular Biology Solutions 

2012 - 2013

supporting 

great science 

through innovation 

in molecular biology 

ABgene 

PCR | qPCR 

Dharmacon 

RNAi | Synthetic RNA 

Fermentas 

Enzymes | Molecular biology tools 

Finnzymes 

PCR | qPCR 

Open Biosystems 

RNAi | Gene Expression 

For years these names represented leading technology, 

reliable results and superior service. Our innovations 

produced novel restriction enzymes, the highest fidelity 

polymerases and most specific siRNAs. Today, the 

people behind our expanding Thermo Scientific research 

portfolio remain committed to supporting your research 

and making it even easier for you to do great science. 

Our Passion – Your Results

II 

Solutions for Molecular Biology 

 

products meet or exceed the industry’s top quality control requirements and provide performance, reliability and 

 

 

 

 

 

 

 

GeneJET Purification 

Kits: 

– Plasmid DNA 

– Genomic DNA 

– Total RNA 

 

An advanced line of 176 restriction enzymes designed for rapid 5-15 minute DNA digestion in the universal FastDigest buffer 

 

Efficient isolation and purification of DNA and RNA from various sources 

 

All-purpose universal DNA ladders for accurate DNA sizing and in-gel quantification 

 

Highly purified dNTPs, NTPs and modified nucleotides for direct use in enzymatic reactions 

 

Fail-safe cloning of PCR products produced with any DNA polymerase 

www.thermoscientific.com/onebio 

 

 

FastDigest 

Restriction Enzymes 

 

 

 

 

 

 

 

 

GeneJET PCR 

Purification Kit 

 

Extraction Kit 

 

 

 

FastAP Alkaline 

Phosphatase 

 

Repair Kit 

 

 

 

 

 

CloneJET PCR 

Cloning Kit 

 

 

 

 

and Expression

In Vitro 

 

 

 

 

 

 

 

 

Find out more about Thermo Scientific products at 


1-233 

235-275 

277-299 

301-323 

325-339 

341-351 

353-383 

385-395 

397-403 

405-417 

419-427 

428-473 

474-495 

www.thermoscientific.com/onebio III

IV 

 

Solutions for Molecular Biology III 

IV 

VIII 

IX 

 

 

 

A new standard in DNA digestion2 

 

 

 

5 

7 

8 

8 

9 

 

72 

72 

72 

72 

72 

 

77 

77 

77 

 

78 

82 

158 

158 

158 

158 

158 

158 

158 

159 

159 

 

 

Recommended Reaction Conditions 161 

165 

Double Digestion in Universal Tango 166 

Digestion of Agarose-Embedded DNA168 

Cleavage Efficiency Close to the Termini of PCR Fragments 169 

 

 

171 

171 

171 

172 

 

 

175 

176 


179 

 

 

 

Compatible DNA Ends 

 

 

 

 

 

 

 

217 

 

 

 

 

 

 

221 

 

222 

 

222 

 

 

 

226 

228 

 

228 

229 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

– 

 

 

251 

252 

Bsm

255 

E.coli 256 

T.maritima 257 

258 

259 

 

261 

262 

 

 

265 

266 

267 

268 

268 

269 

 

 

271 

272 

 

 

 

 

275 

 

 

278 

279 

279 

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282 

285 

286 

287 

288 

289 

 

291 

dam – dcm – 291 

291 

291 

292 

292 

292 

292 

 

 

 

295 

295 

295 

 

295 

296 

E.coli 296 

296 

297 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

mouse tails with Proteinase K 

 

eukaryotic cells with Proteinase K 

 

 

 

diluted solutions with Glycogen 

in vitro transcription 

 

 

 

in vitro 

 

 

 

 

 

 

 

 

 

 


 

In vitro transcription 

 

specific activity 

 

 

 

www.thermoscientific.com/onebio V

VI 

 

 

 

 

 

 

 

 

 

 

 

 


 

 

 

 

 

 

 

GeneRuler 

GeneRuler 

GeneRuler 

GeneRuler 

GeneRuler 

GeneRuler 

GeneRuler 

GeneRuler 

GeneRuler 

GeneRuler 

 

 

 

 

 

 

 

 

 

 

 

FastRuler 

FastRuler 

FastRuler 

FastRuler 

 

 

 

 

 

 

 

 

 

 

 

 


 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

different electrophoresis conditions 

 

 

 

 

 

RiboRuler 

RiboRuler 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

molecular biology grade

molecular biology grade 

 

 

 

 

 


 

 

 

 

6 

4 

5 

 

 

 

 

Transcription Promoter Primers 

18 

 

 

 

Water 

 

 

 

 

 

 

2 

, 

molecular biology grade 

 

, 

molecular biology grade 

 

 

Glycogen, molecular biology grade 

Glycogen, RNA grade 

 

 

 

 

 

 

 

 

 

TopVision Agarose 

TopVision 

 

 

 

E.coli 

 

 

 

X174 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Codons and Assigned Amino Acids 

 

Amino Acids 

 

Physical Constants of the Nucleoside Triphosphates 

and Related Compounds 

 

Common Conversions of Nucleic Acids 

 

 

 

 

General Physical Constants 

Temperature Dependence of the pH for 

 

Decay Factors for Calculating the Amount of Radioactivity 

 

Periodic Table of the Elements 

Commonly Used Abbreviations 

 

 

 

 

 

 

 

www.thermoscientific.com/onebio VII

VIII 

 

 

 

 

international distributors can be found at: 

 

For orders, quotations or any other information, please contact your local 

 

 

 

 

 

 

All products in this catalog have been developed and are sold exclusively 

for research purposes and in vitro 

tested for use in diagnostics or drug development, nor are they suitable 

 

 

 

 

 

 

supplied with a Certificate of Analysis that includes the expiry date of the 

 

However, if a product fails to meet the performance standards stated 

 

 

 

Please note that except for the aforementioned quality warranty, Thermo 

 

or implied, as to the merchantability or fitness of any product for any 

particular use, or non-infringement of third party patent by use of any 

 

 

 

 

To the full extent permitted by applicable law, in no event shall Thermo 

be liable for any direct, indirect, incidental, special, punitive 

 

 

from , the purchaser acknowledges and agrees that 

the sole remedy of any claim by it or for it is free replacement of product 

 

shall hold harmless against any claim resulting 

 


 

The information presented herein is accurate and reliable to the best of our 

 

be construed as recommending any practice or any product in violation of 

 

 

specific purpose and to adopt such safety precautions as may be necessary 

 

 

 

 

mixes, reagents and DNA solutions retain their activity for at least three 

 

 

 

 

 

 

 

 

customers in an on-site freezer program, either in a laboratory or common 

 

 

 

In addition to the product information presented in this product guide, 

you can find the most up-to-date product information in the Certificate of 

Analysis that is provided with each product and at 

 

If you need further help, please do not hesitate to contact your local 

 

 

 

 

scientists will be happy to share the benefit of our experience and 

 

suggestions are always welcome because they help us to better meet

experience enable us to produce the highest quality products for pharmaceutical, molecular 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Development of bacterial expression systems 

In vitro 

Purification of native and recombinant proteins 

Plasmid DNA preparation (pharmaceutical grade meeting FDA and EU guidelines, 

 

RNA production and purification 

Process development 

DNA and RNA ladders 

Processing of nucleic acids by enzymes 

 

DNA and RNA ladders and markers, 

dNTPs, NTPs and modified nucleotides, 

DNA and RNA polymerases, 

 

Restriction enzymes, 

Products for: 

– molecular cloning, 

– nucleic acid purification, 

– protein purification and analysis, 

– molecular labeling and detection, 

– in vitro 

 

 

 

 

 

 

and widely use our unique highest sensitivity tests that are able to assess for trace nuclease 

 

 

 

Products are supplied with a certificate of analysis detailing the conditions and results of quality 

 

 

We welcome your requests by email: 

www.thermoscientific.com/onebio IX

The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC. 

X www.thermoscientific.com/onebio

Thermo Scientific FastDigest & 

Conventional Restriction Enzymes 

FastDigest Restriction Enzymes..................................................................................... 2 

FastDigest and Conventional Restriction Enzymes..................................................... 4 

Quality Control ................................................................................................................. 4 

Labeled Oligonucleotide (LO) Test .................................................................................. 4 

Navigation Guide .............................................................................................................. 5 

Description of Icons ......................................................................................................... 7 

Activity and Quality Control Assays ............................................................................... 8 

Storage and Shipping ...................................................................................................... 8 

Product List ...................................................................................................................... 9 

Product Description ....................................................................................................... 13 

Protocols and Recommendations ................................................................................. 72 

1.1. Fast DNA digestion ................................................................................................ 72 

1.2. Reaction set-up for digestion of multiple DNA samples ........................................... 72 

1.3. Double and multiple digestion of DNA .................................................................... 72 

1.4. Scaling up DNA digestion reaction ......................................................................... 72 

Reaction Conditions ....................................................................................................... 73 

Conventional Restriction Enzymes ............................................................................... 77 

Activity and Quality Control Assays ............................................................................. 77 

Storage and Shipping .................................................................................................... 77 

Product List and Cross-reference to FastDigest Restriction Enzymes ..................... 78 

Product Description ....................................................................................................... 82 

Nicking Enzymes ....................................................................................................... 155 

Homing Enzyme ......................................................................................................... 157 

Protocols and Recommendations ............................................................................... 158 

1.5. DNA digestion .................................................................................................... 158 

1.6. Digestion of PCR products .................................................................................. 158 

1.7. Setting up double digestion ................................................................................. 158 

1.8. Stability during prolonged incubation ................................................................... 158 

1.9. Dilution of restriction enzymes ............................................................................. 158 

1.10. Partial digestion of DNA..................................................................................... 158 

1.11. Digestion of agarose-embedded DNA ................................................................. 159 

1.12. Inactivation of restriction enzymes ..................................................................... 159 

Reaction Conditions ..................................................................................................... 160 

Reaction Buffers ........................................................................................................ 160 

Recommended Reaction Conditions ........................................................................... 161 

Activity of Mesophilic and Thermophilic Enzymes at 37°C ........................................... 165 

Double Digestion in the Universal Tango Buffer ........................................................... 166 

Digestion of Agarose-Embedded DNA ........................................................................ 168 

Cleavage Efficiency Close to the Termini of PCR Fragments ......................................... 169 

Cleavage of Restriction Targets Located in Close Vicinity 

within pUC19 Multiple Cloning Site ............................................................................. 170 

Common Properties ........................................................................................................ 171 

Classification of Restriction Enzymes ....................................................................... 171 

Site Preferences by Restriction Enzymes .................................................................. 171 

Star Activity .................................................................................................................. 172 

Digestion of Methylated DNA ...................................................................................... 173 

Effect of Dam Methylation on DNA Cleavage ................................................................174 

Effect of Dcm Methylation on DNA Cleavage ............................................................... 175 

Effect of CpG Methylation on DNA Cleavage ............................................................... 176 

Effect of EcoKI and EcoBI Methylation on DNA Cleavage ............................................. 179 

Newly Generated Cleavage Sites ................................................................................ 180 

Recognition Sites Resulting from Ligation of Blunt DNA Ends ....................................... 180 

Recognition Sites Resulting from Ligation of Protruding Compatible DNA Ends ............. 190 

Recognition Sites Resulting from Fill-in of 5’-overhang and Self-ligation.......................200 

Recognition Sites Resulting from Removal of 3’-overhang and Self-ligation ..................204 

Selection Guides ..............................................................................................................205 

Alphabetic List of Commercially Available Restriction Enzymes ............................205 

Alphabetic List of Recognition Sequences ................................................................ 217 

Enzyme Recognizing 2 bp Long Targets ....................................................................220 

Alphabetic List of Enzymes Recognizing 4 bp Long Targets ....................................220 


Alphabetic List of Enzymes Recognizing 6 bp Long Targets .................................... 221 



Commercial Restriction Enzymes Generating 5’-protruding Ends ..........................223 

Commercial Restriction Enzymes Generating 3’-protruding Ends ..........................226 

Commercial Restriction Enzymes Generating Blunt Ends ........................................228 

Commercial Restriction Enzymes Cleaving DNA on the Both Sides 

of their Recognition Sequence ....................................................................................228 

Troubleshooting Guide ...................................................................................................229 

Useful webtools: 

www.thermoscientific.com/ReSearch 

www.thermoscientific.com/DoubleDigest 

www.thermoscientific.com/REviewer 

Find out more 

about FastDigest 

Restriction Enzymes: 

www.thermoscientific.com/fd 

www.thermoscientific.com/onebio 1

2 

Thermo Scientific FastDigest Restriction Enzymes 

A new standard in DNA digestion 

Thermo Scientific FastDigest enzymes 

are an advanced line of restriction 

enzymes designed for rapid DNA 

digestion. During our 30 years of 

restriction enzyme research, we 

compiled one of the largest collections 

of restriction enzyme producing 

bacterial strains in the industry. 

Our large selection of enzyme 

isoschizomers and state-of-the-art 

production site facilitated the creation 

of the unique system of 176 FastDigest 

restriction enzymes. All FastDigest 

enzymes are 100% active in the 

universal FastDigest and FastDigest 

Green reaction buffers and are 

able to digest DNA in 5-15 minutes. 

This enables any combination of 

FastDigest restriction enzymes to work 

simultaneously in one tube eliminating 

the need for sequential digestions and 

buffer compatibility charts. As an added 

convenience, the FastDigest Green 

buffer allows for direct loading of the 

reaction mixture on gels. 

FastDigest enzymes are qualified for 

complete digestion of plasmid DNA, 

genomic DNA, viral DNA and PCR 

products. 


Features 

100% activity of all FastDigest enzymes in 

the universal buffer 

100% buffer compatibility with downstream 

applications 

Complete digestion in 5-15 minutes 

Direct loading on gel 

No star activity 

176 FastDigest enzymes available 

30 Years of Restriction Enzyme Research 

and Experience 

 

 

discovered 

 

cloned 

 

 

direct gel loading 

Old Standard 

Conventional restriction 

enzymes 

New Standard 

FastDigest restriction 

enzymes 

Buffer system Up to 20 buffers One universal buffer 

Buffer change between enzymatic steps Frequently necessary Not necessary 

Double/multiple restriction digestion Limited to buffer 

Unlimited – all 176 enzymes 

incompatibility 

are 100% active in the 

universal buffer 

Reaction time for plasmid DNA, 

genomic DNA, PCR product 

1 hour – overnight 5-15 min 

Direct loading of reaction mixture on gels No Yes 

Compatibility with downstream applications Partial 100% 

Star activity Dependent on enzyme 

properties and reaction 

conditions 

Activity definition 1 unit of enzyme hydrolyzes 

1μg of lambda DNA in 60 

minutes in an optimal buffer 

for an enzyme 

Find out 

more at: 

www.thermo 

scientific.com/fd 

No star activity due to short 

incubation time 

1μl of FastDigest enzyme 

cleaves 1μg of substrate 

DNA in 5 or 15 minutes in 

FastDigest buffer

No star activity 

FastDigest enzymes are designed to eliminate 

“star” activity: 

 

the need for prolonged digestions 

 

concentration, 100% compatible with all 176 

FastDigest enzymes 

 

concentrations 

Conventional restriction enzymes may display 

“star” or “relaxed” activity due to prolonged 

incubation times, high enzyme and/or glycerol 

concentration, high pH values or low ionic 

strength. By addressing all these issues, 

FastDigest enzymes enable single, double and 

even triple digestion in 5 minutes without any 

signs of “star” activity. 

100% activity of all FastDigest enzymes 

in the universal buffer: 

 

buffer for any combination of enzymes 

 

changes 

M 1 2 3 4 M 

Five minute triple digestion in the 

FastDigest Green Reaction Buffer 

1. plasmid – DNA undigested control 

2. plasmid – DNA digested with FastDigest EcoRI 

3. plasmid – DNA double digested with FastDigest 

EcoRI and FastDigest KpnI 

4. plasmid – DNA triple digested with FastDigest 

EcoRI, FastDigest KpnI and FastDigest SmaI 

M. marker – Thermo Scientific GeneRuler Express 

Ladder #SM1553 

Visit: 

www.thermo 

scientific.com/ 

REviewer 

100% buffer compatibility with 

downstream applications 

 

FastDigest and FastDigest Green Buffer 

 

be added directly to the restriction digestion 

reaction mixture 

 

steps 

DNA/RNA modifying 

enzyme 

DNA Polymerase I, E.coli 100% 

Klenow Fragment 100% 

Klenow Fragment, exo 100% 

T4 DNA Polymerase 100% 

T7 DNA Polymerase 100% 

T4 DNA Ligase* 75-100% 

FastAP Thermosensitive 

Alkaline Phosphatase 

Activity in 

FastDigest Green 

Buffer/FastDigest 

Buffer 

100% 

T4 Polynucleotide Kinase 100% 

* 0.5 mM ATP is required for T4 DNA Ligase activity 

Useful web 

tool REsearch: 

Find restriction 

enzymes either by name 

or recognition 

sequence 

Useful web 

tool REviewer: 

DNA sequence analysis, 

plasmid map creation 

and biochemical 

calculations 

Direct loading on gels 

The 10X FastDigest Green Buffer includes 

a density reagent and two tracking dyes for 

direct loading of digestion reaction products 

on gels. The blue dye migrates with 3-5 kb 

DNA fragments in a 1% agarose gel and has 

an excitation peak of 424 nm. The yellow dye 

migrates faster than 10 bp DNA fragments in 

a 1% agarose gel and has an excitation peak 

of 615 nm. 

Visit: 

www.thermo 

scientific.com/ 

REsearch 

1 2 

FastDigest Green Buffer 

Reaction mixture containing 

FastDigest Green Buffer: 

1. Loaded into gel well, 

before electrophoresis 

2. Resolved in the gel, after 

electrophoresis 

3-5 kb 

10 bp 


4 

FastDigest and Conventional Restriction Enzymes 

Restriction enzymes recognize specific 

nucleotide sequences and cleave DNA 

molecules at a position either within or outside 

their recognition site. These enzymes are 

important tools for numerous applications, 

including studies of DNA primary structure and 

recombinant DNA technology. The best studied 

and the most widely used are type II restriction 

endonucleases. More than 3800 type II 

restriction enzymes, exhibiting almost 290 

Quality Control 

Thermo Scientific Restriction enzymes are 

produced in clean room class D facilities under 

the ISO 9001:2008 quality management 

system and are subjected to extensive 

quality control. 

Thermo Scientific restriction enzymes 

pass all industry standard quality control 

assays, as well as the unique Labeled 

 

most sensitive test for the detection of 

trace activities of endodeoxyribonucleases, 

exodeoxyribonucleases and phosphatases 

see 

A warranty is assigned and an expiry date 

is listed both on the product label and in the 

Certificate of Analysis supplied with each 

product. Product lots are monitored to meet 

the quality specifications up to the expiry date. 

Labeled Oligonucleotide (LO) 

Test 

The Labeled Oligonucleotide Test was designed 

for detection of trace contaminating activities 

in enzyme, nucleotide and reagent solutions. 

This unique test allows us to detect trace 

activities of endo-, exodeoxyribonucleases 

and phosphatases in enzyme preparations. 

Therefore we are able to bring to the market 

only highest quality and performance products. 

Single-stranded and double-stranded 

5’-[ 32 P]-labeled synthetic oligonucleotides 

containing no recognition sites for restriction 

enzymes are used as substrates for the LO 

test. The labeled oligonucleotides are incubated 

with excess of restriction enzyme, separated 

on a polyacrylamide gel under denaturing 

conditions and analyzed by phospho-imaging. 


different specificities, have been isolated. 

Since 1977, we have discovered approximately 

30% of all known restriction enzymes. We are a 

leading global enzyme manufacturer, supplying 

both conventional restriction enzymes and 

innovative restriction enzyme line for rapid DNA 

digestion in one universal buffer – FastDigest 

restriction enzymes. We offer 176 FastDigest 

and 190 conventional restriction enzymes. 

The presence of contaminating endo- and 

exodeoxyribonucleases results in the degradation 

of the labeled substrates, while decreased 

specific radioactivity indicates the presence 

of contaminant phosphatases. The restriction 

enzyme passes this quality control test if there is 

neither degradation of labeled oligonucleotides 

nor a decrease in specific radioactivity. 

Labeled Oligonucleotide (LO) Test. 

ss – single-stranded radiolabeled oligonucleotide 

ds – double-stranded radiolabeled oligonucleotide 

Pure enzyme – Thermo Scientific NotI 

Contaminated enzyme – competitor’s NotI 

Labeled Oligonucleotide Test 

Radiolabeled ss and ds oligonucleotides 

mixed with an excess of enzyme 

Control 

Incubated at 37°C for 4 hours 

Denatured and separated by PAGE 

Band pattern imaged and analyzed 

Patterns Typical for: 

Pure 

enzyme 

Contaminated 

enzyme 

ss ds ss ds ss ds 

Degradation due 

to contamination 

with non-specific 

nucleases

Navigation Guide 

Table 1.1. Thermo Scientific FastDigest and conventional restriction enzymes. 

To find or select a restriction enzyme Page 

By name 

By recognition sequence 

By number of recognition sites 

in DNA 

By type of generated DNA ends 

By cleavage efficiency close to 

DNA termini 

By sensitivity to DNA methylation 

By newly generated recognition 

sites 

Table 1.2. Thermo Scientific FastDigest restriction enzymes 2 

Table 1.4. Thermo Scientific conventional restriction enzymes 78 

Table 1.25. Commercially available restriction enzymes 205 

On-line. REsearch at www.thermoscientific.com/research 

Table 1.26. Recognition sequences of restriction enzymes 217 

Table 1.27. Enzyme recognizing 2 bp long targets 220 

Table 1.28. Enzymes recognizing 4 bp long targets 220 






Table 12.15. Number of recognition sites in DNA molecules 457 

Appendix. Phage and plasmid DNA 432 

On-line. REviewer at www.thermoscientific.com/reviewer 

Table 1.33. Commercial restriction enzymes generating 5’-protruding ends 223 

Table 1.34. Commercial restriction enzymes generating 3’-protruding ends 226 

Table 1.35. Commercial restriction enzymes generating blunt ends 228 

Table 1.36. Commercial restriction enzymes cleaving DNA on the both sides of their recognition sequence 228 


Table 1.3. Reaction conditions for Thermo Scientific FastDigest restriction enzymes 73 

Table 1.10. Cleavage efficiency close to the termini of PCR fragments 169 

Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site 170 

Table 1.12. Thermo Scientific isoschizomers and neoschizomers with differing sensitivities to target methylation 173 

Table 1.13, Table 1.14. Effect of Dam methylation 174 

Table 1.15, Table 1.16. Effect of Dcm methylation 175 

Table 1.17, Table 1.18. Effect of CpG methylation 176 

Table 1.19, Table 1.20. Effect of EcoBI and EcoKI methylation 179 

Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends 180 

Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends 190 

Table 1.23. Newly generated recognition sites resulting from fill-in of 5’-overhang and self-ligation 200 

Table 1.24. Newly generated recognition sites resulting from removal of 3’-overhang and self-ligation 204 


6 

To perform fast digestion of DNA in universal FastDigest buffer 

Fast DNA digestion reaction set-up Protocols and recommendations for fast DNA digestion 72 

Digestion of different DNA substrates 

Digestion close to DNA termini 

Inactivation of enzyme after digestion 


Table 1.3. Reaction conditions for Thermo Scientific FastDigest restriction enzymes 73 

Incubation time without star activity 

Compatibility of FastDigest enzyme buffer with downstream 

applications 

2.1. Activity of DNA/RNA modifying enzymes in Thermo Scientific buffers 274 

Troubleshooting Table 1.37. Troubleshooting guide for DNA digestion 230 

To perform DNA digestion with conventional restriction enzymes 

DNA digestion reaction set-up Protocols and recommendations for DNA digestion with conventional restriction enzymes 158 

Double/multiple digestion of DNA 

Reaction conditions for individual enzymes 

Digestion close to DNA termini 

On-line. DoubleDigest at www.thermoscientific.com/doubledigest 

Table 1.8. Double digestion in the universal Tango buffer 166 

Table 1.6. Reaction conditions for restriction enzymes 161 


Table 1.5. Reaction buffers for restriction enzymes 160 

Table 1.10. Cleavage efficiency close to the termini of PCR fragments 169 

Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site 170 

Digestion of agarose-embeded DNA 1.11 Table 1.9. Digestion of agarose-embedded DNA 159 

Inactivation of enzyme after digestion 

1.12. Inactivation of restriction enzymes 159 


How to identify and avoid star activity Description of star activity of restriction enzymes 172 

Compatibility of conventional restriction enzyme buffers 

with downstream applications 

2.1. Activity of DNA/RNA modifying enzymes in Thermo Scientific buffers 274 

Partial digestion of DNA 1.10. Partial digestion of DNA 158 

How to dilute restriction enzymes 1.9. Dilution of restriction enzymes 158 

Stability during prolonged incubation Table 1.6. Reaction conditions for restriction enzymes 161 

Enzyme activity at non-optimal temperature Table 1.7. Activity of mesophilic and thermophilic enzymes at 37°C 165 

Troubleshooting guide Table 1.37. Troubleshooting guide for DNA digestion 230

Description of Icons 

15’ 

20’ 

Single letter code 

R = G or A; H = A, C or T; 

Y = C or T; V = A, C or G; 

W = A or T; B = C, G or T; 

M = A or C; D = A, G or T; 

K = G or T; N = G, A, T or C. 

S = C or G; 

FastDigest Enzyme. Enzyme is available in FastDigest format see 

Buffers. Letters in the buffer icon indicate the recommended buffer for a specific restriction 

enzyme. FastDigest and FastDigest 

 

conventional restriction enzymes. The “Unique” icon indicates conventional restriction enzymes that 

require a special buffer, which is supplied with the enzyme see 

Additives. Indicates additives required to obtain the stated enzyme activity. Solutions of 

 

 

Incubation Temperature. 

Star Activity. see 

Sensitivity to Methylation. DNA cleavage by the restriction enzyme is blocked or impaired by 

Dam, Dcm or CpG methylation within the target sequences see 

Sensitivity to Overlapping Dam, Dcm or CpG Methylation. The target site may be methylated 

 

cleavage see 

Thermal Inactivation. see 

Thermal Inactivation. 

can be thermally inactivated. 

High Concentration. . 

Genome Qualified. see 

Recombinant Enzyme. Indicates enzymes purified from recombinant E.coli. 

LO Certified. Enzyme was tested by the see 

NEW for 2012. 


8 

Activity and Quality 

Control Assays 

Activity Assay 

1 μl of FastDigest enzyme is formulated to 

cleave 1 μg of substrate DNA in 5 or 15 min at 

recommended temperature in 1X FastDigest or 

1X FastDigest Green buffers. 1 μl of FastDigest 

restriction endonuclease corresponds to 1 

FastDigest 

In general, enzymes are assayed with phage 

DNA at 37°C. However, some exceptions apply: 

 

activity at temperatures other than 37°C are 

assayed under their optimal temperature. 

enzymes that do not have 

recognition sites on DNA are assayed with 

other specific DNA substrates, as indicated in 

the Certificates of Analysis. 

enzymes with only a few 

recognition sites on the DNA are assayed 

using DNA cut with another restriction 

enzyme. 

enzymes sensitive to Dam or Dcm 

methylation are assayed using DNA purified 

from dam – , dcm – strain of E.coli. 


Labeled Oligonucleotide (LO) Test 

1μl of FastDigest enzyme is incubated 

with 5’-[ 32P]-labeled single-stranded and 

double-stranded synthetic oligonucleotides 

in 1X FastDigest buffer for 1 hour at the 

recommended temperature. The restriction 

enzyme passes this quality control test if there 

is no degradation of labeled oligonucleotides 

and no decrease in their specific radioactivity. 

Blue/White (B/W) Cloning Assay 

The B/W assay was replaced by LO test after 

validating experiments showed LO test ability to 

detect nuclease and phosphatase activities with 

sensitivity that equals to that of B/W test. 


Ligation and Recleavage (L/R) Assay 

The ligation and recleavage assay was replaced 

with LO test after validating experiments 

revealed LO test ability to trace nuclease and 

phosphatase activities with sensitivity that is 

higher than L/R test by a factor of 100. 

Prolonged Incubation / 

Star Activity Assay 

The star activity assay is designed to test 

alterations in the DNA digestion pattern after 

prolonged incubation with FastDigest enzymes. 

1 μl of FastDigest enzyme is incubated with 

1 μg of substrate DNA at the recommended 

temperature for up to 16 hours. After 

electrophoretic separation of the DNA 

fragments, the characteristic banding patterns 

are examined for alterations. The maximal 

incubation time that does not cause any star 

activity is indicated for each FastDigest enzyme 

in the product description and the Certificate of 

Analysis supplied with each enzyme. 

Storage and Shipping 

All FastDigest restriction enzymes should be 

stored at -20°C. 

During shipment on dry ice, enzymes may 

freeze. This does not affect their quality – all 

Thermo Scientific restriction enzymes are 100% 

active after at least three freeze-thaw cycles. 

For 24-48 hour delivery, enzymes may be 

shipped on blue ice since their quality is not 

affected by short exposure to 4°C.

Product List 

Table 1.2. FastDigest restriction enzymes. 

FastDigest 

restriction enzyme 

Prototype Specificity 5’3’ Cat. # Page 

FastDigest AatII AatII GACGTC FD0994 13 

FastDigest AccI AccI GTMKAC FD1484 13 

FastDigest Acc65I GGTACC FD0904 13 

FastDigest AciI AciI FD1794 14 

FastDigest AclI AclI AACGTT FD0944 14 

FastDigest AcuI Eco57I FD0344 14 

FastDigest AfeI Eco47III AGCGCT FD0324 15 

FastDigest AflII AflII CTTAAG FD0834 15 

FastDigest AgeI AgeI ACCGGT FD1464 15 

FastDigest AjuI AjuI 7 FD1954 16 

FastDigest AleI OliI CACNNNNGTG FD1634 16 

FastDigest AluI AluI AGCT FD0014 16 

FastDigest Alw21I HgiAI GWGCWC FD0024 17 

FastDigest Alw26I BsmAI FD0034 17 

FastDigest AlwNI AlwNI CAGNNNCTG FD1394 17 

FastDigest ApaI ApaI GGGCCC FD1414 18 

FastDigest ApaLI ApaLI GTGCAC FD0044 18 

FastDigest AscI AscI GGCGCGCC FD1894 18 

FastDigest AseI VspI ATTAAT FD0914 19 

FastDigest AsiSI SgfI GCGATCGC FD2094 19 

FastDigest AvaI AvaI CYCGRG FD0384 19 

FastDigest AvaII AvaII GGWCC FD0314 20 

FastDigest AvrII AvrII CCTAGG FD1564 20 

FastDigest BamHI BamHI GGATCC FD0054/5 20 

FastDigest BanI HgiCI GGYRCC FD1004 21 

FastDigest BbsI BbvII FD1014 21 

FastDigest BbvI BbvI FD2074 21 

FastDigest BclI BclI TGATCA FD0724 22 

FastDigest BfaI MaeI CTAG FD1764 22 

FastDigest BglI BglI GCCNNNNNGGC FD0074 22 

FastDigest BglII BglII AGATCT FD0083/4 23 

FastDigest BlpI EspI GCTNAGC FD0094 23 

FastDigest Bme1580I BseSI GKGCMC FD1444 23 

FastDigest BmtI GCTAGC FD2044 24 

FastDigest BplI BplI 5 FD1314 24 

FastDigest BpmI BpmI FD0464 24 

FastDigest Bpu10I Bpu10I FD1184 25 

FastDigest BsaAI BsaAI YACGTR FD1974 25 

FastDigest BsaBI BsaBI GATNNNNATC FD1414 25 

FastDigest BsaHI AcyI GRCGYC FD0474 26 

FastDigest BsaJI SecI CCNNGG FD1084 26 

FastDigest BseGI FD0874 26 

FastDigest BseNI BsrI FD0884 27 

FastDigest BseXI BbvI FD1454 27 

FastDigest Bsh1236I FnuDII CGCG FD0924 27 

 


10 

Table 1.2. Thermo Scientific FastDigest restriction enzymes. 

FastDigest 



FastDigest BsiEI McrI CGRYCG FD0894 28 

FastDigest BsiWI SplI CGTACG FD0854 28 

FastDigest BslI BsiYI CCNNNNNNNGG FD1204 28 

FastDigest BsmBI Esp3I FD0454 29 

FastDigest BsmFI FinI FD1814 29 

FastDigest Bsp119I AsuII TTCGAA FD0124 29 

FastDigest Bsp120I GGGCCC FD0134 30 

FastDigest Bsp1286I SduI GDGCHC FD0654 30 

FastDigest Bsp1407I Bsp1407I TGTACA FD0933/4 30 

FastDigest BspCNI BseMII FD1404 31 

FastDigest BspHI BspHI TCATGA FD1284 31 

FastDigest BspMI BspMI FD1744 31 

FastDigest BsrBI BsrBI FD1274 32 

FastDigest BsrDI BsrDI FD1264 32 

FastDigest BsrFI Cfr10I RCCGGY FD0184 32 

FastDigest BssHII BsePI GCGCGC FD2134 33 

FastDigest BstXI BstXI CCANNNNNNTGG FD1024 33 

FastDigest BstZ17I SnaI GTATAC FD0704 33 

FastDigest Bsu36I SauI CCTNAGG FD0374 34 

FastDigest ClaI ClaI ATCGAT FD0143/4 34 

FastDigest Csp6I GTAC FD0214 34 

FastDigest DdeI DdeI CTNAG FD1884 34 

FastDigest DpnI DpnI Gm6ATC FD1703/4 35 

FastDigest DraI AhaIII TTTAAA FD0224 35 

FastDigest DraIII DraIII CACNNNGTG FD1234 36 

FastDigest DrdI DrdI GACNNNNNNGTC FD1724 36 

FastDigest EagI XmaIII CGGCCG FD0334 36 

FastDigest Eam1105I Eam1105I GACNNNNNGTC FD0244 37 

FastDigest EarI Ksp632I FD0234 37 

FastDigest Ecl136II GAGCTC FD0254 37 

FastDigest Eco31I Eco31I FD0293/4 38 

FastDigest Eco91I BstEII GGTNACC FD0394 38 

FastDigest EcoNI EcoNI CCTNNNNNAGG FD1304 38 

FastDigest EcoO109I DraII RGGNCCY FD0264 39 

FastDigest EcoRI EcoRI GAATTC FD0274/5 39 

FastDigest EcoRV EcoRV GATATC FD0303/4 39 

FastDigest EheI GGCGCC FD0443/4 40 

FastDigest Fnu4HI Fnu4HI GCNGC FD1644 40 

FastDigest FokI FokI FD2144 40 

FastDigest FspI MstI TGCGCA FD1224 41 

FastDigest FspAI FspAI RTGCGCAY FD1664 41 

FastDigest HaeII HaeII RGCGTY FD2184 41 

FastDigest HaeIII HaeIII GGCC FD0154 42 

FastDigest HgaI HgaI FD1904 42 

FastDigest HhaI HhaI GCGC FD1854 42 

FastDigest HincII HindII GTYRAC FD0494 43 

FastDigest HindIII HindIII AAGCTT FD0504/5 43 

FastDigest HinfI HinfI GANTC FD0804 43 

FastDigest HinP1I GCGC FD0484 44 

FastDigest HpaI HpaI GTTAAC FD1034 44 

www.thermoscientific.com/onebio


FastDigest 



FastDigest HpaII HpaII CCGG FD0514 44 

FastDigest Hpy8I MjaIV GTNNAC FD1574 45 

FastDigest HpyF10VI MwoI GCNNNNNNNGC FD1734 45 

FastDigest KpnI KpnI GGTACC FD0524 45 

FastDigest Kpn2I BspMII TCCGGA FD0534 46 

FastDigest MauBI MauBI CGCGCGCG FD2084 46 

FastDigest MboI MboI GATC FD0814 46 

FastDigest MboII MboII FD0824 47 

FastDigest MfeI MfeI CAATTG FD0753/4 47 

FastDigest MluI MluI ACGCGT FD0564 47 

FastDigest MlyI FD1374 48 

FastDigest MnlI MnlI FD1074 48 

FastDigest MreI Sse232I CGCCGGCG FD2024 48 

FastDigest MscI BalI TGGCCA FD1214 49 

FastDigest MseI MseI TTAA FD2174 49 

FastDigest MslI MslI CAYNNNNRTG FD2004 49 

FastDigest MspI HpaII CCGG FD0544 50 

FastDigest MssI PmeI GTTTAAAC FD1344 50 

FastDigest MvaI CCWGG FD0554 50 

FastDigest Mva1269I BsmI FD0964 51 

FastDigest NaeI NaeI GCCGGC FD1524 51 

FastDigest NciI CauII CCSGG FD0064 51 

FastDigest NcoI NcoI CCATGG FD0573/4/5 52 

FastDigest NdeI NdeI CATATG FD0583/4/5 52 

FastDigest NheI NheI GCTAGC FD0973/4 52 

FastDigest NlaIII NlaIII CATG FD1834 53 

FastDigest NlaIV NlaIV GGNNCC FD1154 53 

FastDigest NmuCI Tsp45I GTSAC FD1514 53 

FastDigest NotI NotI GCGGCCGC FD0593/4/6 54 

FastDigest NruI NruI TCGCGA FD2154 54 

FastDigest NsiI AvaIII ATGCAT FD0734 54 

FastDigest NspI NspI RCATGY FD1474 55 

FastDigest PacI PacI TTAATTAA FD2204 55 

FastDigest PdmI XmnI GAANNNNTTC FD1534 55 

FastDigest PflMI PflMI CCANNNNNTGG FD0714 56 

FastDigest PfoI PfoI TCCNGGA FD1754 56 

FastDigest PmlI PmaCI CACGTG FD0364 56 

FastDigest PpuMI PpuMI RGGWCCY FD0764 57 

FastDigest PshAI PshAI GACNNNNGTC FD1434 57 

FastDigest PsiI PsiI TTATAA FD2064 57 

FastDigest PspFI BseYI FD2224 58 

FastDigest PstI PstI CTGCAG FD0614/5 58 

FastDigest PsuI XhoII RGATCY FD1554 58 

FastDigest PsyI Tth111I GACNNNGTC FD1334 59 

FastDigest PvuI PvuI CGATCG FD0624 59 

FastDigest PvuII PvuII CAGCTG FD0634 59 

FastDigest RsaI RsaI GTAC FD1124 60 

FastDigest RsrII RsrII CGGWCCG FD0744 60 

 


12 


FastDigest 



FastDigest SacI SacI GAGCTC FD1133/4 60 

FastDigest SalI SalI GTCGAC FD0644 61 

FastDigest SanDI SanDI GGGWCCC FD2164 61 

FastDigest SapI SapI FD1934 61 

FastDigest Sau3AI MboI GATC FD0784 62 

FastDigest Sau96I AsuI GGNCC FD0194 62 

FastDigest SbfI Sse8387I CCTGCAGG FD1194 62 

FastDigest ScaI ScaI AGTACT FD0434 63 

FastDigest ScrFI ScrFI CCNGG FD1424 63 

FastDigest SexAI SexAI ACCWGGT FD2114 63 

FastDigest SfaNI SfaNI FD2124 64 

FastDigest SfcI SfeI CTRYAG FD1164 64 

FastDigest SfiI SfiI GGCCNNNNNGGCC FD1824 64 

FastDigest SmaI SmaI CCCGGG FD0663/4 65 

FastDigest SnaBI SnaBI TACGTA FD0404 65 

FastDigest SpeI SpeI ACTAGT FD1253/4 65 

FastDigest SphI SphI GCATGC FD0604 66 

FastDigest SspI SspI AATATT FD0774 66 

FastDigest StuI StuI AGGCCT FD0424 66 

FastDigest StyI StyI CCWWGG FD0414 67 

FastDigest SwaI SwaI ATTTAAAT FD1244 67 

FastDigest TaaI Tsp4CI ACNGT FD1364 67 

FastDigest TaiI ACGT FD1144 68 

FastDigest TaqI TaqI TCGA FD0674 68 

FastDigest TatI TatI WGTACW FD1294 68 

FastDigest TauI TauI GCSGC FD1654 69 

FastDigest TfiI TfiI GAWTC FD1784 69 

FastDigest Tru1I MseI TTAA FD0984 69 

FastDigest Tsp509I TspEI AATT FD1354 70 

FastDigest TspRI TspRI FD2104 70 

FastDigest XapI ApoI RAATTY FD1383/4 70 

FastDigest XbaI XbaI TCTAGA FD0684/5 71 

FastDigest XhoI XhoI CTCGAG FD0694/5 71 







S = C or G; 


Product Description 

FastDigest AatII 

5’...G A C G TC...3’ 

3’...C T G C A G...5’ 

#FD0994 50 μl 

Supplied with: 

10X FastDigest Buffer 1 ml 

10X FastDigest Green Buffer 1 ml 

pUC19DNA/SmaI, 1.0% agarose 

1 cleavage site 

Formulation 

 

DNA/SmaI fragments in 15 min at 37°C in 

1X FastDigest Buffer. 

Reaction Conditions 

1X FastDigest Buffer or 1X FastDigest Green 

Buffer at 37°C. 

Methylation Effects 

Dam, Dcm, EcoKI, EcoBI – no effect. 

CpG: completely overlaps – blocked 

Digestion time with 1 μl of FastDigest AatII bp from end of DNA required Thermal Incubation time without 

Lambda DNA, 1μg/20μl Plasmid DNA, 1μg/20μl PCR product, ~0.2μg/30μl Genomic DNA, 1μg/10μl for complete digestion inactivation star activity 

15 min 20 min 15 min 15 min 5 bp 80°C, 5 min 16 hours 

FastDigest AccI 

5’...G TM K A C...3’ 

3’...C A K M T G...5’ 

#FD1484 50 μl 




 

DNA, 1.0% agarose 

9 cleavage sites 

Formulation 

 

DNA in 5 min at 37°C in 1X FastDigest Buffer. 






CpG: may overlap – blocked 

Digestion time with 1 μl of FastDigest AccI bp from end of DNA required Thermal Incubation time without 



FastDigest Acc65I 

5’...GG T A C C...3’ 

3’...C C A T G G...5’ 

#FD0904 100 μl 




 



Formulation 

 

DNA/BamHI fragments in 5 min at 37°C in 






Dam, EcoKI, EcoBI – no effect. 

Dcm, CpG: may overlap – cleavage impaired 

 

Note 

Acc65I cleavage is impaired by 

overlapping dcm methylation. To avoid dcm 

methylation, use a dam – , dcm – strain such 

as GM2163. 

Digestion time with 1 μl of FastDigest Acc65I bp from end of DNA required Thermal Incubation time without 




14 

FastDigest AciI 

5’...CC G C...3’ 

3’...G G C G...5’ 

#FD1794 20 μl 




Digestion time with 1 μl of FastDigest AciI bp from end of DNA required Thermal Incubation time without 




 



FastDigest AclI 

5’...A AC G T T...3’ 

3’...T T G C A A...5’ 

#FD0944 20 μl 






Formulation 

 





Formulation 

 









Dam, Dcm – no effect. 


EcoKI, EcoBI: may overlap – effect not 

determined. 

Digestion time with 1 μl of FastDigest AclI bp from end of DNA required Thermal Incubation time without 



FastDigest AcuI 

5’...C T G A A G (N) 16...3’ 

3’...G A C T T C (N) 14 ...5’ 

#FD0344 20 μl 




 

 



Formulation 

 





SAM 0.01 mM. 


Dam, Dcm, CpG, EcoKI – no effect. 

EcoBI: may overlap – effect not determined. 

Note 

 

Mg 2+ for its activity, but is stimulated by 

S-adenosylmethionine. Still, complete 

cleavage of some substrates with 

FastDigest 

achieve. 

 

at least two copies of its recognition 

sequence are required. 

 

associated with the cleaved DNA. This 

may cause DNA band shifting during 

electrophoresis. To avoid atypical DNA 

band patterns heat the digested DNA in 

the presence of 6X DNA Loading Dye & 

 

electrophoresis. 

Digestion time with 1 μl of FastDigest AcuI bp from end of DNA required Thermal Incubation time without 


15 min 30 min 15 min 15 min 2 bp 65°C, 5 min 16 hours

FastDigest AfeI 

5’...A G CG C T...3’ 

3’...T C G C G A...5’ 

#FD0324 20 μl 




 



Formulation 

 

DNA/Eco81I fragments in 5 min at 37°C in 






Dam, Dcm, EcoKI – no effect. 



Digestion time with 1 μl of FastDigest AfeI bp from end of DNA required Thermal Incubation time without 



FastDigest AflII 

5’...CT T A A G...3’ 

3’...G A A T T C...5’ 

#FD0834 150 μl 




 



Formulation 

 







Dam, Dcm, CpG, EcoBI, EcoKI – no effect. 

Digestion time with 1 μl of FastDigest AflII bp from end of DNA required Thermal Incubation time without 


5 min 5 min 5 min 5 min 4 bp No 16 hours 

FastDigest AgeI 

5’...AC C G G T...3’ 

3’...T G G C C A...5’ 

#FD1464 20 μl 




 



Formulation 

 








EcoKI, EcoBI: may overlap – effect not 

determined. 

Digestion time with 1 μl of FastDigest AgeI bp from end of DNA required Thermal Incubation time without 




16 

FastDigest AjuI 

5’...7(N) G A A(N) 7 T T G G (N) 11...3’ 

3’... 12(N) C T T(N) 7 A A C C (N) 6 ...5’ 

#FD1954 20 μl 




 


X174DNA, 1.0% agarose 


Formulation 

X174 





SAM 0.01 mM. 


Dam, Dcm, CpG, EcoKI, EcoBI – no effect. 

Note 

AjuI requires 

S-adenosylmethionine for activity. Still, 

complete cleavage of some substrates with 

FastDigest AjuI is difficult to achieve. 

AjuI produces double-strand cuts 

on both sides of the interrupted recognition 

site. In certain sequence contexts, the 

cleavage position may be shifted by one 

base pair. However, the cleavage position 

indicated above will predominate. 

Digestion time with 1 μl of FastDigest AjuI bp from end of DNA required Thermal Incubation time without 


5 min 5 min 10 min 5 min ND 65°C, 5 min 16 hours 

FastDigest AleI 

5’...C A C N NN N G T G...3’ 

3’...G T G N N N N C A C...5’ 

#FD1634 20 μl 




X174DNA, 1.0% agarose 


Formulation 

X174 







CpG: may overlap – cleavage impaired 

EcoKI, EcoBI: may overlap – blocked 

Digestion time with 1 μl of FastDigest AleI bp from end of DNA required Thermal Incubation time without 



FastDigest AluI 

5’...A GC T...3’ 

3’...T C G A...5’ 

#FD0014 100 μl 




 



Formulation 

 







EcoBI: may overlap – blocked 

Digestion time with 1 μl of FastDigest AluI bp from end of DNA required Thermal Incubation time without 



FastDigest Alw21I 

5’...G W G C WC...3’ 

3’...C W C G W G...5’ 

#FD0024 100 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest Alw21I bp from end of DNA required Thermal Incubation time without 




5’...G T C T C(N) 1...3’ 

3’...C A G A G(N) 5 ...5’ 

#FD0034 100 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest Alw26I bp from end of DNA required Thermal Incubation time without 



FastDigest AlwNI 

5’...C A G N N NC T G...3’ 

3’...G T C N N N G A C...5’ 

#FD1394 50 μl 




 



Formulation 

 






Dam, CpG, EcoKI – no effect. 

Dcm: may overlap – blocked 


Note 

FastDigest 

dcm methylation. To avoid dcm methylation, use a 

dam – , dcm – strain such as GM2163. 

Digestion time with 1 μl of FastDigest AlwNI bp from end of DNA required Thermal Incubation time without 




18 

FastDigest ApaI 

5’...G G G C CC...3’ 

3’...C C C G G G...5’ 

#FD1414 300 μl 




Digestion time with 1 μl of FastDigest ApaI bp from end of DNA required Thermal Incubation time without 




 



FastDigest ApaLI 

5’...GT G C A C...3’ 

3’...C A C G T G...5’ 

#FD0044 200 μl 




 



Formulation 

 

DNA/CpoI fragments in 5 min at 37°C in 





Formulation 

 









 

Note 

ApaI cleavage is impaired by 

overlapping dcm methylation. To avoid 

dcm methylation, use a dam – , dcm – strain 

such as GM2163. 




Digestion time with 1 μl of FastDigest ApaLI bp from end of DNA required Thermal Incubation time without 



FastDigest AscI 

5’...G GC G C G C C...3’ 

3’...C C G C G C G G...5’ 

#FD1894 100 μl 




 



Formulation 

 






Digestion time with 1 μl of FastDigest AscI bp from end of DNA required Thermal Incubation time without 



20’ 



CpG: completely overlaps – blocked

FastDigest AseI 

5’...A TT A A T...3’ 

3’...T A A T T A...5’ 

#FD0914 200 μl 




 

DNA , 1.0% agarose 


Formulation 

 








Digestion time with 1 μl of FastDigest AseI bp from end of DNA required Thermal Incubation time without 



FastDigest AsiSI 

5’...G C G A TC G C...3’ 

3’...C G C T A G C G...5’ 

#FD2094 100 μl 




 


Formulation 

 

 

5 min at 37°C in 1X FastDigest Buffer. 







Digestion time with 1 μl of FastDigest AsiSI bp from end of DNA required Thermal Incubation time without 


no cleavage sites 5 min 60 min 5 min 5 bp 80°C, 5 min 6 hours 

FastDigest AvaI 

5’...CY C G R G...3’ 

3’...G R G C Y C...5’ 

#FD0384 200 μl 




 



Formulation 

 







CpG: completely overlaps – 

cleavage impaired 

Digestion time with 1 μl of FastDigest AvaI bp from end of DNA required Thermal Incubation time without 




20 

FastDigest AvaII 

5’...GG W C C ...3’ 

3’...C C W G G...5’ 

#FD0314 200 μl 




Digestion time with 1 μl of FastDigest AvaII bp from end of DNA required Thermal Incubation time without 




 


FastDigest AvrII 

5’...CC T A G G...3’ 

3’...G G A T C C...5’ 

#FD1564 20 μl 




 




Formulation 

 





Formulation 

 








Dcm, CpG: may overlap – blocked 

Note 

 



as GM2163. 



Digestion time with 1 μl of FastDigest AvrII bp from end of DNA required Thermal Incubation time without 



FastDigest BamHI 

5’...GG A T C C...3’ 

3’...C C T A G G...5’ 

#FD0054 800 μl 


10X FastDigest buffer 2x1 ml 

10X FastDigest Green Buffer 2x1 ml 

#FD0055 2500 μl 




 



Formulation 

 

DNA/Bsp120I fragments in 5 min at 37°C in 







Digestion time with 1 μl of FastDigest BamHI bp from end of DNA required Thermal Incubation time without 


5 min 5 min 5 min 5 min 2 bp 80°C, 5 min 1 hour

FastDigest BanI 

5’...GG Y R C C...3’ 

3’...C C R Y G G...5’ 

#FD1004 300 μl 




 

– 


Formulation 

 

DNA dcm – in 5 min at 37°C in 1X FastDigest 

Buffer. 





Dam, EcoBI – no effect. 


 

EcoKI: may overlap – effect not determined. 

Note 

 

by overlapping dcm methylation. To avoid 



Digestion time with 1 μl of FastDigest BanI bp from end of DNA required Thermal Incubation time without 



FastDigest BbsI 

5’...G A A G A C(N) 2...3’ 

3’...C T T C T G(N) 6 ...5’ 

#FD1014 20 μl 




 

DNA. 0.7% agarose 


Formulation 

 








Digestion time with 1 μl of FastDigest BbsI bp from end of DNA required Thermal Incubation time without 



FastDigest BbvI 

5’...G C A G C (N) 8 ...3’ 

3’...C G T C G (N) 12 ...5’ 

#FD2074 100 μl 




pBR322DNA, 0.7% agarose 


Formulation 

 








Digestion time with 1 μl of FastDigest BbvI bp from end of DNA required Thermal Incubation time without 


5 min 5 min 5 min 10 min 2 bp 65°C, 5 min 0.5 hour 


22 

FastDigest BclI 

5’...TG A T C A...3’ 

3’...A C T A G T...5’ 

#FD0724 300 μl 




Digestion time with 1 μl of FastDigest BclI bp from end of DNA required Thermal Incubation time without 




 

dam – 

FastDigest BfaI 

5’...CT A G...3’ 

3’...G A T C...5’ 

#FD1764 50 μl 




 




Formulation 

 

DNA dam – in 5 min at 37°C in 1X FastDigest 

Buffer. 




Formulation 

 






Dam: completely overlaps – blocked 

Dcm, CpG, EcoKI – no effect. 


Note 

BclI is blocked by dam 

methylation. To avoid dam methylation, use 

a dam – , dcm – strain such as GM2163. 



Digestion time with 1 μl of FastDigest BfaI bp from end of DNA required Thermal Incubation time without 



FastDigest BglI 

5’...G C C N N N NN G G C...3’ 

3’...C G G N N N N N C C G...5’ 

#FD0074 200 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest BglI bp from end of DNA required Thermal Incubation time without 



FastDigest BglII 

5’...AG A T C T...3’ 

3’...T C T A G A...5’ 

#FD0083 100 μl 

#FD0084 200 μl 

Both supplied with: 



 



Formulation 

 







EcoBI: may overlap – cleavage impaired 

Digestion time with 1 μl of FastDigest BglII bp from end of DNA required Thermal Incubation time without 



FastDigest BlpI 

5’...G CT N A G C...3’ 

3’...C G A N T C G...5’ 

#FD0094 50 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest BlpI bp from end of DNA required Thermal Incubation time without 



FastDigest Bme1580I 

5’...G K G C MC...3’ 

3’...C M C G K G...5’ 

#FD1444 50 μl 




 



Formulation 

 






Dam, Dcm, CpG, EcoBI – no effect. 

EcoKI: may overlap – cleavage impaired 

Digestion time with 1 μl of FastDigest Bme1580I bp from end of DNA required Thermal Incubation time without 




24 

FastDigest BmtI 

5’...G C T A GC...3’ 

3’...C G A T C G...5’ 

#FD2044 20 μl 




Digestion time with 1 μl of FastDigest BmtI bp from end of DNA required Thermal Incubation time without 


5 min 5 min 15 min 5 min 3 bp 80°C, 5 min 1 hour 

FastDigest BplI 


 


5’...8(N) G A G(N) 5 C T C(N) 13...3’ 

3’... 13(N) C T C(N) 5 G A G(N) 8 ...5’ 

#FD1314 20 μl 




 

 

DNA/XhoI, 0.7% agarose 



Formulation 

 

DNA/HindIII fragments in 5 min at 37°C in 





Formulation 

 

DNA/XhoI fragments in 15 min at 37°C in 





SAM 0.05 mM. 





Note 

2 + 

for its activity, but is stimulated by 


cleavage of some substrates with 

FastDigest BplI is difficult to achieve. 

Digestion time with 1 μl of FastDigest BplI bp from end of DNA required Thermal Incubation time without 


15 min 20 min 30 min 30 min ND 65°C, 5 min 16 hours 

FastDigest BpmI 

5’...C T G G A G (N) 16...3’ 

3’...G A C C T C (N) 14 ...5’ 

#FD0464 20 μl 




 



Formulation 

 






Dam, CpG, EcoKI, EcoBI – no effect. 


Note 

 


S-adenosylmethionine. 

 



as GM2163. 

Digestion time with 1 μl of FastDigest BpmI bp from end of DNA required Thermal Incubation time without 



FastDigest Bpu10I 

5’...C CT N A G C...3’ 

3’...G G A N T C G...5’ 

#FD1184 20 μl 




M13mp18 DNA, 0.7% agarose 


Formulation 

 

M13mp18 DNA in 15 min at 37°C in 







Note 

 

two copies of its recognition sequence are 

required. 

Digestion time with 1 μl of FastDigest Bpu10I bp from end of DNA required Thermal Incubation time without 



FastDigest BsaAI 

5’...Y A CG T R...3’ 

3’...R T G C A Y...5’ 

#FD1974 50 μl 




 



Formulation 

 








EcoKI, EcoBI: may overlap – effect not determined. 

Digestion time with 1 μl of FastDigest BsaAI bp from end of DNA required Thermal Incubation time without 



FastDigest BsaBI 

5’...G A T N NN N A T C...3’ 

3’...C T A N N N N T A G...5’ 

#FD1714 200 μl 




 

dam – 


Formulation 

 


Buffer. 





Dam: may overlap – blocked 

Dcm, EcoKI, CpG – no effect. 


Note 

 

overlapping dam methylation. To avoid dam 


as GM2163. 

Digestion time with 1 μl of FastDigest BsaBI bp from end of DNA required Thermal Incubation time without 


5 min 5 min 5 min 5 min 3 bp No 1 hour 


26 

FastDigest BsaHI 

5’...G RC G Y C...3’ 

3’...C Y G C R G...5’ 

#FD0474 100 μl 




Digestion time with 1 μl of FastDigest BsaHI bp from end of DNA required Thermal Incubation time without 




 

– 

FastDigest BsaJI 

5’...CC N N G G...3’ 

3’...G G N N C C...5’ 

#FD1084 20 μl 




 




Formulation 

 


Buffer. 




Formulation 

 






Dam, EcoKI – no effect. 

Dcm: may overlap – cleavage impaired 



Note 

 

impaired by overlapping dcm methylation. 

To avoid dcm methylation, use a dam – , dcm – 

strain such as GM2163. 



Digestion time with 1 μl of FastDigest BsaJI bp from end of DNA required Thermal Incubation time without 



FastDigest BseGI 

5’...G G A T G N N...3’ 

3’...C C T A C N N ...5’ 

#FD0874 100 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest BseGI bp from end of DNA required Thermal Incubation time without 



FastDigest BseNI 

5’...A C T G G N...3’ 

3’...T G A C C N ...5’ 

#FD0884 100 μl 




 



Formulation 

 






Dam, Dcm, CpG – no effect. 


Digestion time with 1 μl of FastDigest BseNI bp from end of DNA required Thermal Incubation time without 



FastDigest BseXI 

5’...G C A G C(N) 8 ...3’ 

3’...C G T C G(N) 12 ...5’ 

#FD1454 20 μl 




pBR322 DNA, 1.4% agarose 


Formulation 

 








Digestion time with 1 μl of FastDigest BseXI bp from end of DNA required Thermal Incubation time without 



FastDigest Bsh1236I 

5’...C GC G...3’ 

3’...G C G C...5’ 

#FD0924 100 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest Bsh1236I bp from end of DNA required Thermal Incubation time without 




28 

FastDigest BsiEI 

5’...C G R YC G...3’ 

3’...G C Y R G C...5’ 

#FD0894 100 μl 




Digestion time with 1 μl of FastDigest BsiEI bp from end of DNA required Thermal Incubation time without 




 


FastDigest BsiWI 

5’...CG T A C G...3’ 

3’...G C A T G C...5’ 

#FD0854 50 μl 




 




Formulation 

 





Formulation 

 

DNA/Psp1406I fragments in 15 min at 37°C in 






Dam: may overlap – cleavage impaired 

Dcm, EcoKI, EcoBI – no effect. 


Note 

 

impaired by overlapping dam methylation. 

To avoid dam methylation, use a dam – , dcm – 





Digestion time with 1 μl of FastDigest BsiWI bp from end of DNA required Thermal Incubation time without 



FastDigest BslI 

5’...C C N N N N NN N G G...3’ 

3’...G G N N N N N N N C C...5’ 

#FD1204 100 μl 




 

– 


Formulation 

 


Buffer. 







 

Note 

 

by overlapping dcm methylation. To avoid 



Digestion time with 1 μl of FastDigest BslI bp from end of DNA required Thermal Incubation time without 


5 min 5 min 5 min 5 min 2 bp No 16 hours

FastDigest BsmBI 

5’...C G T C T C(N) 1...3’ 

3’...G C A G A G(N) 5 ...5’ 

#FD0454 20 μl 




 


14 clavage sites 

Formulation 

 





 





Note 

 

Freshly made DTT solution should be added 

to the reaction buffer. 

Digestion time with 1 μl of FastDigest BsmBI bp from end of DNA required Thermal Incubation time without 



FastDigest BsmFI 

5’...G G G A C(N) 10...3’ 

3’...C C C T G(N) 14 ...5’ 

#FD1814 20 μl 




 

 



Formulation 

 





SAM 0.05 mM. 


Dam, Dcm, EcoBI, EcoKI – no effect. 


Note 

 



cleavage of some substrates is difficult to 

achieve. 

 

least two copies of its recognition sequence 

are required. 

Digestion time with 1 μl of FastDigest BsmFI bp from end of DNA required Thermal Incubation time without 



FastDigest Bsp119I 

5’...T TC G A A...3’ 

3’...A A G C T T...5’ 

#FD0124 200 μl 




 



Formulation 

 






Dam, Dcm, EcoBI – no effect. 



Digestion time with 1 μl of FastDigest Bsp119I bp from end of DNA required Thermal Incubation time without 




30 


5’...GG G C C C...3’ 

3’...C C C G G G...5’ 

#FD0134 200 μl 







5’...G D G C HC...3’ 

3’...C H C G D G...5’ 

#FD0654 50 μl 





 



FastDigest Bsp1286I 

 



Formulation 

 






Formulation 

 








Note 

Bsp120I is blocked by 



as GM2163. 








5’...TG T A C A...3’ 

3’...A C A T G T...5’ 

#FD0933 50 μl 

#FD0934 100 μl 




 



Formulation 

 










FastDigest BspCNI 

5’...C T C A G(N) 10...3’ 

3’...G A G T C(N) 8 ...5’ 

#FD1404 20 μl 




 

 



Formulation 

 





SAM 0.01 mM. 


Dam, Dcm, CpG, EcoBI, EcoKI – no effect. 

Note 

 

S-adenosylmethionine for activity. Sinefungin 

can replace SAM in the restriction reaction. 

In this case DNA is not methylated and more 

 

fragments can be recut by this enzyme. 

Digestion time with 1 μl of FastDigest BspCNI bp from end of DNA required Thermal Incubation time without 



FastDigest BspHI 

5’...TC A T G A...3’ 

3’...A G T A C T...5’ 

#FD1284 50 μl 




X174 DNA, 1.0% agarose 


Formulation 

X174 







Dam, EcoBI: may overlap – cleavage impaired 

 

Note 

 

by overlapping dam methylation. To avoid 

dam methylation, use a dam – , dcm – strain 


Digestion time with 1 μl of FastDigest BspHI bp from end of DNA required Thermal Incubation time without 



FastDigest BspMI 

5’...A C C T G C(N) 4...3’ 

3’...T G G A C G(N) 8 ...5’ 

#FD1744 50 μl 




 

 


Note 

BspMI 

 

efficient cleavage. 

 

the FastDigest 


Formulation 

 





Oligonucleotide 0.5 μM. 

sequence in the reaction mixture 

significantly improves cleavage of plasmid 

DNAs, especially of those with a single 

FastDigest site. Still, complete 

cleavage of some substrates is difficult to 

achieve. 




EcoBI, EcoKI: may overlap – effect not determined. 

 






 


Digestion time with 1 μl of FastDigest BspMI bp from end of DNA required Thermal Incubation time without 




32 

FastDigest BsrBI 

5’...C C GC T C...3’ 

3’...G G C G A G...5’ 

#FD1274 100 μl 




Digestion time with 1 μl of FastDigest BsrBI bp from end of DNA required Thermal Incubation time without 



5’...G C A A T G N N...3’ 

3’...C G T T A C N N ...5’ 

#FD1264 20 μl 





 


FastDigest BsrDI 

 




Formulation 

 





Formulation 

 













Digestion time with 1 μl of FastDigest BsrDI bp from end of DNA required Thermal Incubation time without 



FastDigest BsrFI 

5’...RC C G G Y...3’ 

3’...Y G G C C R...5’ 

#FD0184 50 μl 




 



Formulation 

 









Note 

 

at least two copies of its recognition 

sequence are required. 

Digestion time with 1 μl of FastDigest BsrFI bp from end of DNA required Thermal Incubation time without 



FastDigest BssHII 

5’...GC G C G C...3’ 

3’...C G C G C G...5’ 

#FD2134 20 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest BssHII bp from end of DNA required Thermal Incubation time without 



FastDigest BstXI 

5’...C C A N N N N NN T G G...3’ 

3’...G G T N N N N N N A C C...5’ 

#FD1024 100 μl 

SUPPLIED WITH: 



 



Formulation 

 







Dcm: may overlap – cleavage impaired 

Note 

BstXI cleavage is impaired by 



as GM2163. 

Digestion time with 1 μl of FastDigest BstXI bp from end of DNA required Thermal Incubation time without 


5 min 5 min 5 min 5 min 4 bp 80°C, 5 min 0.5 hours 

FastDigest BstZ17I 

5’...G T AT A C...3’ 

3’...C A T A T G...5’ 

#FD0704 100 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest BstZ17I bp from end of DNA required Thermal Incubation time without 




34 

FastDigest Bsu36I 

5’...C CT N A G G...3’ 

3’...G G A N T C C...5’ 

#FD0374 50 μl 




Digestion time with 1 μl of FastDigest Bsu36I bp from end of DNA required Thermal Incubation time without 



5’...A TC G A T...3’ 

3’...T A G C T A...5’ 

#FD0143 50 μl 

#FD0144 100 μl 





 


FastDigest ClaI 

 




Formulation 

 






Formulation 

 











Dcm, EcoKI – no effect. 


Note 

 

overlapping dam methylation. To avoid dcm 


as GM2163. 

Digestion time with 1 μl of FastDigest ClaI bp from end of DNA required Thermal Incubation time without 



FastDigest Csp6I 

5’...GT A C...3’ 

3’...C A T G...5’ 

#FD0214 100 μl 




 



Formulation 

 







Digestion time with 1 μl of FastDigest Csp6I bp from end of DNA required Thermal Incubation time without 



FastDigest DdeI 

5’...CT N A G...3’ 

3’...G A N T C...5’ 

#FD1884 50 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest DdeI bp from end of DNA required Thermal Incubation time without 



FastDigest DpnI 

5’...G m6A T C...3’ 

3’...C T m6A G...5’ 

#FD1703 50 μl 

#FD1704 100 μl 






Formulation 

 

 





Note 

DpnI requires the presence of 

N6-methyladenine within the recognition 

sequence to cleave DNA. 

dam + strain will be a 

substrate for FastDigest DpnI. 


Dam: does not cut dam – DNA 



DpnI will only cleave fully-adenomethylated 

dam sites. 

DpnI, FastDigest Sau3AI 

MboI all recognize 

the same sequence but have different 

 

Digestion time with 1 μl of FastDigest DpnI bp from end of DNA required Thermal Incubation time without 


not determined 5 min not applicable 10 min 1 bp 80°C, 5 min 16 hours 

FastDigest DraI 

5’...T T TA A A...3’ 

3’...A A A T T T...5’ 

#FD0224 200 μl 




 



Formulation 

 







EcoKI: may overlap – blocked 

Digestion time with 1 μl of FastDigest DraI bp from end of DNA required Thermal Incubation time without 




36 

FastDigest DraIII 

5’...C A C N N NG T G...3’ 

3’...G T G N N N C A C...5’ 

#FD1234 50 μl 




Digestion time with 1 μl of FastDigest DraIII bp from end of DNA required Thermal Incubation time without 



FastDigest EagI 

5’...CG G C C G...3’ 

3’...G C C G G C...5’ 

#FD0334 50 μl 




Digestion time with 1 μl of FastDigest EagI bp from end of DNA required Thermal Incubation time without 




 


FastDigest DrdI 

5’...G A C N N N NN N G T C...3’ 

3’...C T G N N N N N N C A G...5’ 

#FD1724 25 μl 




 


 




Formulation 

 





Formulation 

 





Formulation 

 













Digestion time with 1 μl of FastDigest DrdI bp from end of DNA required Thermal Incubation time without 







FastDigest Eam1105I 

5’...G A C N N NN N G T C...3’ 

3’...C T G N N N N N C A G...5’ 

#FD0244 100 μl 




FastDigest EarI 

5’...C T C T T C(N) 1...3’ 

3’...G A G A A G(N) 4 ...5’ 

#FD0234 50 μl 




Digestion time with 1 μl of FastDigest EarI bp from end of DNA required Thermal Incubation time without 



FastDigest Ecl136II 

5’...G A GC T C...3’ 

3’...C T C G A G...5’ 

#FD0254 100 μl 




 


X174 DNA, 1.0% agarose 

 



Digestion time with 1 μl of FastDigest Eam1105I bp from end of DNA required Thermal Incubation time without 





Formulation 

 





Formulation 

X174 





Formulation 

 











Note 

DNA are difficult to cleave 

with FastDigest 

as with its prototype Ksp632I. 





Digestion time with 1 μl of FastDigest Ecl136II bp from end of DNA required Thermal Incubation time without 




38 

FastDigest Eco31I 

5’...G G T C T C(N) 1...3’ 

3’...C C A G A G(N) 5 ...5’ 

#FD0293 50 μl 

#FD0294 100 μl 




Digestion time with 1 μl of FastDigest Eco31I bp from end of DNA required Thermal Incubation time without 




5’...GG T N A C C...3’ 

3’...C C A N T G G...5’ 

#FD0394 200 μl 





 

dcm – 

 




Formulation 

 

DNA dcm – /HindIII fragments in 5 min at 37°C in 





Formulation 

 








 


Note 

Eco31I cleavage is impaired by 



as GM2163. 




Digestion time with 1 μl of FastDigest Eco91I bp from end of DNA required Thermal Incubation time without 



FastDigest EcoNI 

5’...C C T N NN N N A G G...3’ 

3’...G G A N N N N N T C C...5’ 

#FD1304 100 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest EcoNI bp from end of DNA required Thermal Incubation time without 



FastDigest EcoO109I 

5’...R GG N C C Y...3’ 

3’...Y C C N G G R...5’ 

#FD0264 200 μl 




 


3 clevage sites 

Formulation 

 










Note 

EcoO109I is blocked by 


methylation, use a dam – , dcm – strain such as 

GM2163. 

Digestion time with 1 μl of FastDigest EcoO109I bp from end of DNA required Thermal Incubation time without 



FastDigest EcoRI 

5’...GA A T T C...3’ 

3’...C T T A A G...5’ 

#FD0274 800 μl 




#FD0275 2500 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest EcoRI bp from end of DNA required Thermal Incubation time without 



FastDigest EcoRV 

5’...G A TA T C...3’ 

3’...C T A T A G...5’ 

#FD0303 200 μl 

#FD0304 400 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest EcoRV bp from end of DNA required Thermal Incubation time without 




40 

FastDigest EheI 

5’...G G CG C C...3’ 

3’...C C G C G G...5’ 

#FD0443 20 μl 

#FD0444 50 μl 




Digestion time with 1 μl of FastDigest EheI bp from end of DNA required Thermal Incubation time without 



5’...G CN G C...3’ 

3’...C G N C G...5’ 

#FD1644 20 μl 





 


FastDigest Fnu4HI 

 




Formulation 

 

DNA/PstI fragments in 5 min at 37°C in 





Formulation 

 











Note 

Fnu4HI 

 


Digestion time with 1 μl of FastDigest Fnu4HI bp from end of DNA required Thermal Incubation time without 



FastDigest FokI 

5’...G G A T G(N) 9...3’ 

3’...C C T A C(N) 13 ...5’ 

#FD2144 100 μl 




 



Formulation 

 








Note 

FokI 

recognition site are required for an efficient 

cleavage. 

FokI may remain associated 

with the cleaved DNA. This may cause DNA 

band shifting during electrophoresis. To 

avoid atypical DNA band patterns heat the 

digested DNA in the presence of 6X DNA 

 

SDS prior to electrophoresis. 

Digestion time with 1 μl of FastDigest FokI bp from end of DNA required Thermal Incubation time without 



FastDigest FspI 

5’...T G CG C A...3’ 

3’...A C G C G T...5’ 

#FD1224 50 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest Fsp bp from end of DNA required Thermal Incubation time without 



FastDigest FspAI 

5’...R T G CG C A Y...3’ 

3’...Y A C G C G T R...5’ 

#FD1664 20 μl 




 



Formulation 

 

DNA/Psp1406I fragments in 5 min at 37°C in 









Digestion time with 1 μl of FastDigest FspAI bp from end of DNA required Thermal Incubation time without 



FastDigest HaeII 

5’...R G C G CY...3’ 

3’...Y C G C G R...5’ 

#FD2184 200 μl 




 



Formulation 

 








EcoBI: may overtlap – not determined. 

Digestion time with 1 μl of FastDigest HaeII bp from end of DNA required Thermal Incubation time without 




42 

FastDigest HaeIII 

5’...G GC C...3’ 

3’...C C G G...5’ 

#FD0154 400 μl 




Digestion time with 1 μl of FastDigest HaeIII bp from end of DNA required Thermal Incubation time without 



5’...G A C G C(N) 5...3’ 

3’...C T G C G(N) 10 ...5’ 

#FD1904 20 μl 





 


FastDigest HgaI 




Formulation 

 





Formulation 

 






Dam, Dcm, EcoKI: never overlaps – no effect. 





Note 

 


are required. 

 






 


Digestion time with 1 μl of FastDigest HgaI bp from end of DNA required Thermal Incubation time without 



FastDigest HhaI 

5’...G C GC...3’ 

3’...C G C G...5’ 

#FD1854 200 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest HhaI bp from end of DNA required Thermal Incubation time without 



FastDigest HincII 

5’...G T YR A C...3’ 

3’...C A R Y T G...5’ 

#FD0494 100 μl 




 



Formulation 

 








EcoKI, EcoBI: may overlap – blocked 

Digestion time with 1 μl of FastDigest HincII bp from end of DNA required Thermal Incubation time without 



FastDigest HindIII 

5’...AA G C T T...3’ 

3’...T T C G A A...5’ 

#FD0504 800 μl 




#FD0505 2500 μl 




 



Formulation 

 







EcoBI: may overlap – cleavage impaired 

Digestion time with 1 μl of FastDigest HindIII bp from end of DNA required Thermal Incubation time without 



FastDigest HinfI 

5’...GA N T C...3’ 

3’...C T N A G...5’ 

#FD0804 400 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest HinfI bp from end of DNA required Thermal Incubation time without 



20’ 


44 

FastDigest HinP1I 

5’...GC G C...3’ 

3’...C G C G...5’ 

#FD0484 200 μl 




Digestion time with 1 μl of FastDigest HinP1I bp from end of DNA required Thermal Incubation time without 



5’...G T TA A C...3’ 

3’...C A A T T G...5’ 

#FD1034 50 μl 





 


FastDigest HpaI 

 




Formulation 

 





Formulation 

 












Digestion time with 1 μl of FastDigest HpaI bp from end of DNA required Thermal Incubation time without 



FastDigest HpaII 

5’...CC G G...3’ 

3’...G G C C...5’ 

#FD0514 200 μl 




 



Formulation 

 





Digestion time with 1 μl of FastDigest HpaII bp from end of DNA required Thermal Incubation time without 



20’ 




FastDigest Hpy8I 

5’...G T NN A C...3’ 

3’...C A N N T G...5’ 

#FD1574 20 μl 




 



Formulation 

 






Dam, Dcm, – no effect. 


EcoKI, EcoBI – effect not determined. 

Digestion time with 1 μl of FastDigest Hpy8I bp from end of DNA required Thermal Incubation time without 



FastDigest HpyF10VI 

5’...G C N N N N NN N G C...3’ 

3’...C G N N N N N N N C G...5’ 

#FD1734 20 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest HpyF10VI bp from end of DNA required Thermal Incubation time without 



FastDigest KpnI 

5’...G G T A CC...3’ 

3’...C C A T G G...5’ 

#FD0524 300 μl 




 



Formulation 

 







Dam, Dcm, CpG, EcoKI, EcoKI, EcoBI – no effect. 

Digestion time with 1 μl of FastDigest KpnI bp from end of DNA required Thermal Incubation time without 




46 

FastDigest Kpn2I 

5’...TC C G G A...3’ 

3’...A G G C C T...5’ 

#FD0534 100 μl 




Digestion time with 1 μl of FastDigest Kpn2I bp from end of DNA required Thermal Incubation time without 



FastDigest MauBI 

5’...C GC G C G C G...3’ 

3’...G C G C G C G C...5’ 

#FD2084 20 μl 





 


 



Formulation 

 





Formulation 

 

 

recognition sites in 5 min at 37°C in 











Digestion time with 1 μl of FastDigest MauBI bp from end of DNA required Thermal Incubation time without 



FastDigest MboI 

5’...G A T C ...3’ 

3’... C T A G ...5’ 

#FD0814 50 μl 




 

dam – 


Formulation 

 


Buffer. 








Note 

MboI is blocked by overlapping 

dam methylation. To avoid dam methylation, 

use a dam – , dcm – strain such as GM2163. 

MboI, FastDigest Sau3AI 

DpnI all recognize 


 

Digestion time with 1 μl of FastDigest MboI bp from end of DNA required Thermal Incubation time without 



FastDigest MboII 

5’...G A A G A(N) 8...3’ 

3’...C T T C T(N) 7 ...5’ 

#FD0824 50 μl 




 

dam – 


Formulation 

 


Buffer. 






Dcm, CpG, EcoKI, EcoBI – no effect. 

Note 

MboII is blocked by overlapping 



MboII produces DNA fragments 

that have a single-base 3’-extension which 

are more difficult to ligate than blunt-ended 

fragments. 

MboII may remain associated 





 


Digestion time with 1 μl of FastDigest MboII bp from end of DNA required Thermal Incubation time without 



FastDigest MfeI 

5’...CA A T T G...3’ 

3’...G T T A A C...5’ 

#FD0753 20 μl 

#FD0754 50 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest MfeI bp from end of DNA required Thermal Incubation time without 



FastDigest MluI 

5’...AC G C G T...3’ 

3’...T G C G C A...5’ 

#FD0564 100 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest MluI bp from end of DNA required Thermal Incubation time without 




48 

FastDigest MlyI 

5’...G A G T C(N) 5...3’ 

3’...C T C A G(N) 5 ...5’ 

#FD1374 100 μl 




Digestion time with 1 μl of FastDigest Mly bp from end of DNA required Thermal Incubation time without 



FastDigest MnlI 

5’...C C T C(N) 7...3’ 

3’...G G A G(N) 6 ...5’ 

#FD1074 50 μl 





 


 




Formulation 

 





Formulation 

 











Note 

MnlI produces DNA fragments 

that have a single-base 3’-extension which 

are more difficult to ligate than blunt-ended 

fragments. 

Digestion time with 1 μl of FastDigest MnlI bp from end of DNA required Thermal Incubation time without 



FastDigest MreI 

5’...C GC C G G C G...3’ 

3’...G C G G C C G C...5’ 

#FD2024 20 μl 




 


Formulation 

 

 








Digestion time with 1 μl of FastDigest MreI bp from end of DNA required Thermal Incubation time without 


no cleavage sites 5 min 5 min 5 min 3 bp 80°C, 5 min 16 hours

FastDigest MscI 

5’...T G GC C A...3’ 

3’...A C C G G T...5’ 

#FD1214 50 μl 




 

dcm – 


Formulation 

 


Buffer. 







Note 

FastDigest 

dcm methylation. To avoid dcm methylation, use a 


Digestion time with 1 μl of FastDigest MscI bp from end of DNA required Thermal Incubation time without 



FastDigest MseI 

5’...TT A A...3’ 

3’...A A T T...5’ 

#FD2174 50 μl 






Formulation 

 








Digestion time with 1 μl of FastDigest MseI bp from end of DNA required Thermal Incubation time without 



FastDigest MslI 

5’...C A Y N NN N R T G...3’ 

3’...G T R N N N N Y A C...5’ 

#FD2004 20 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest MslI bp from end of DNA required Thermal Incubation time without 



20’ 


50 

FastDigest MspI 

5’...CC G G...3’ 

3’...G G C C...5’ 

#FD0544 400 μl 




Digestion time with 1 μl of FastDigest MspI bp from end of DNA required Thermal Incubation time without 



FastDigest MssI 

5’...G T T TA A A C...3’ 

3’...C A A A T T T G...5’ 

#FD1344 100 μl 





 


 




Formulation 

 





Formulation 

 








Note 

MspI is an isoschizomer of 

HpaII. When the external C in the sequence 

CCGG is methylated, these enzymes cannot 

cleave. However, unlike HpaII, FastDigest 

MspI can cleave the sequence when the 

internal C residue is methylated. 




Digestion time with 1 μl of FastDigest MssI bp from end of DNA required Thermal Incubation time without 



FastDigest MvaI 

5’...C CW G G...3’ 

3’...G G W C C...5’ 

#FD0554 200 μl 




 



Formulation 

 







Digestion time with 1 μl of FastDigest MvaI bp from end of DNA required Thermal Incubation time without 


5 min 5 min 5 min 10 min 4 bp No 1 hour

FastDigest Mva1269I 

5’...G A A T G C N...3’ 

3’...C T T A C G N ...5’ 

#FD0964 50 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest Mva1269I bp from end of DNA required Thermal Incubation time without 



FastDigest NaeI 

5’...G C CG G C...3’ 

3’...C G G C C G...5’ 

#FD1524 50 μl 




pBR322 DNA/NdeI, 0.7% agarose 


Formulation 

 

DNA/NdeI fragments in 5 min at 37°C in 








Note 

 


are required. 

 

cleave with FastDigest 

as with its prototype NaeI. 

Digestion time with 1 μl of FastDigest NaeI bp from end of DNA required Thermal Incubation time without 


not determined 5 min 5 min 5 min 3 bp 65°C, 15 min 16 hours 

FastDigest NciI 

5’...C CS G G...3’ 

3’...G G S C C...5’ 

#FD0064 200 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest NciI bp from end of DNA required Thermal Incubation time without 




52 

FastDigest NcoI 

5’...CC A T G G...3’ 

3’...G G T A C C...5’ 

#FD0573 20 μl 

#FD0574 100 μl 

#FD0575 300 μl 

All supplied with: 



Digestion time with 1 μl of FastDigest NcoI bp from end of DNA required Thermal Incubation time without 



FastDigest NdeI 

5’...C AT A T G...3’ 

3’...G T A T A C...5’ 

#FD0583 100 μl 

#FD0584 300 μl 




#FD0585 1000 μl 


10X FastDigest Buffer 2X1 ml 

10X FastDigest Green Buffer 2X1 ml 


 


 




Formulation 

 





Formulation 

 









Digestion time with 1 μl of FastDigest NdeI bp from end of DNA required Thermal Incubation time without 



FastDigest NheI 

5’...GC T A G C...3’ 

3’...C G A T C G...5’ 

#FD0973 50 μl 

#FD0974 100 μl 




 

DNA 0.7% agarose 


Formulation 

 









Digestion time with 1 μl of FastDigest NheI bp from end of DNA required Thermal Incubation time without 



FastDigest NlaIII 

5’... C A T G ...3’ 

3’... G T A C ...5’ 

#FD1834 100 μl 




 



Formulation 

 








Note 

 

the half-life of FastDigest 

months. 

Digestion time with 1 μl of FastDigest NlaIII bp from end of DNA required Thermal Incubation time without 



FastDigest NlaIV 

5’...G G NN C C...3’ 

3’...C C N N G G...5’ 

#FD1154 20 μl 




 



Formulation 

 








 

Digestion time with 1 μl of FastDigest NlaIV bp from end of DNA required Thermal Incubation time without 



FastDigest NmuCI 

5’...G T S A C ...3’ 

3’... C A S T G ...5’ 

#FD1514 20 μl 




 



Formulation 

 





20’ 

Note 

 

impaired by overlapping dcm methylation. 







Digestion time with 1 μl of FastDigest NmuCI bp from end of DNA required Thermal Incubation time without 




54 

FastDigest NotI 

5’...G CG G C C G C...3’ 

3’...C G C C G G C G...5’ 

#FD0593 20 μl 

#FD0594 50 μl 

#FD0596 250 μl 




Digestion time with 1 μl of FastDigest NotI bp from end of DNA required Thermal Incubation time without 



FastDigest NruI 

5’...T C GC G A...3’ 

3’...A G C G C T...5’ 

#FD2154 100 μl 





 

 




Formulation 

 

 

37°C in 1X FastDigest Buffer. 




Formulation 

 











Digestion time with 1 μl of FastDigest NruI bp from end of DNA required Thermal Incubation time without 



FastDigest NsiI 

5’...A T G C AT...3’ 

3’...T A C G T A...5’ 

#FD0734 100 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest NsiI bp from end of DNA required Thermal Incubation time without 



FastDigest NspI 

5’...R C A T GY...3’ 

3’...Y G T A C R...5’ 

#FD1474 50 μl 




 



Formulation 

 






Dam, Dcm, EcoKI, EcoBI, CpG – no effect. 

Digestion time with 1 μl of FastDigest NspI bp from end of DNA required Thermal Incubation time without 



FastDigest PacI 

5’...T T A A TT A A...3’ 

3’...A A T T A A T T...5’ 

#FD2204 25 μl 




 


Formulation 

 

 

PacI recognition site in 5 min at 37°C in 






Dam, Dcm, EcoBI, CpG – no effect. 


Digestion time with 1 μl of FastDigest PacI bp from end of DNA required Thermal Incubation time without 



FastDigest PdmI 

5’...G A A N NN N T T C...3’ 

3’...C T T N N N N A A G...5’ 

#FD1534 100 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest PdmI bp from end of DNA required Thermal Incubation time without 




56 

FastDigest PflMI 

5’...C C A N N N NN T G G...3’ 

3’...G G T N N N N N A C C...5’ 

#FD0714 100 μl 




Digestion time with 1 μl of FastDigest PflMI bp from end of DNA required Thermal Incubation time without 



FastDigest PfoI 

5’...TC C N G G A...3’ 

3’...A G G N C C T...5’ 

#FD1754 20 μl 





 


 




Formulation 

 





Formulation 

 








Note 

 



as GM2163. 


Dam: may overlap – cleavage impaired 


EcoKI, EcoBI – no effect. 

Note 

PfoI cleavage is impaired by 

overlapping dam methylation and blocked by 

overlapping dcm methylation. To avoid dam 

or dcm methylation, use a dam – , dcm – strain 


Digestion time with 1 μl of FastDigest PfoI bp from end of DNA required Thermal Incubation time without 



FastDigest PmlI 

5’...C A CG T G...3’ 

3’...G T G C A C...5’ 

#FD0364 200 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest PmlI bp from end of DNA required Thermal Incubation time without 



FastDigest PpuMI 

5’...R GG W C C Y...3’ 

3’...Y C C W G G R...5’ 

#FD0764 50 μl 




 



Formulation 

 










Note 

 



as GM2163. 

Digestion time with 1 μl of FastDigest PpuMI bp from end of DNA required Thermal Incubation time without 



FastDigest PshAI 

5’...G A C N NN N G T C...3’ 

3’...C T G N N N N C A G...5’ 

#FD1434 100 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest PshAI bp from end of DNA required Thermal Incubation time without 



FastDigest PsiI 

5’...T T AT A A ...3’ 

3’...A A T A T T ...5’ 

#FD2064 20 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest Psi bp from end of DNA required Thermal Incubation time without 




58 

FastDigest PspFI 

5’...C C C A GC...3’ 

3’...G G G T C G...5’ 

#FD2224 20 μl 




Digestion time with 1 μl of FastDigest PspFI bp from end of DNA required Thermal Incubation time without 


5 min 5 min 5 min 5 min 4 80°C, 5 min 16 hours 

FastDigest PstI 

5’...C T G C AG...3’ 

3’...G A C G T C...5’ 

#FD0614 800 μl 




#FD0615 2500 μl 





 


 




Formulation 

 





Formulation 

 










Note 

 

adjacent runs of G-C base pairs confers 

significant resistance to cleavage 

 

 

PstI will not cut AGCTGCAG when 

methylated by AluI methyltransferase. 

Digestion time with 1 μl of FastDigest PstI bp from end of DNA required Thermal Incubation time without 



FastDigest PsuI 

5’...RG A T C Y...3’ 

3’...Y C T A G R...5’ 

#FD1554 150 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest PsuI bp from end of DNA required Thermal Incubation time without 



FastDigest PsyI 

5’...G A C NN N G T C...3’ 

3’...C T G N N N C A G...5’ 

#FD1334 100 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest PsyI bp from end of DNA required Thermal Incubation time without 



FastDigest PvuI 

5’...C G A TC G...3’ 

3’...G C T A G C...5’ 

#FD0624 20 μl 




FastDigest PvuII 

5’...C A GC T G...3’ 

3’...G T C G A C...5’ 

#FD0634 400 μl 




 


Digestion time with 1 μl of FastDigest PvuI bp from end of DNA required Thermal Incubation time without 



 




Formulation 

 






Formulation 

 











Digestion time with 1 μl of FastDigest PvuII bp from end of DNA required Thermal Incubation time without 


5 min 5 min 5 min 5 min 1 bp No 0.5 hour 


60 

FastDigest RsaI 

5’...G TA C...3’ 

3’...C A T G...5’ 

#FD1124 100 μl 




Digestion time with 1 μl of FastDigest RsaI bp from end of DNA required Thermal Incubation time without 



5’...C GG W C C G...3’ 

3’...G C C W G G C...5’ 

#FD0744 20 μl 





 


FastDigest RsrII 

 




Formulation 

 





Formulation 

 











Digestion time with 1 μl of FastDigest RsrII bp from end of DNA required Thermal Incubation time without 



FastDigest SacI 

5’...G A G C TC...3’ 

3’...C T C G A G...5’ 

#FD1133 100 μl 

#FD1134 200 μl 




 



Formulation 

 








Note 

SacI is sensitive to cytosine 

methylation at GAGmCTC but not GAGCTmC 

and insensitive to adenine methylation at 

 

can be used to block FastDigest SacI. 

Digestion time with 1 μl of FastDigest SacI bp from end of DNA required Thermal Incubation time without 



FastDigest SalI 

5’...GT C G A C...3’ 

3’...C A G C T G...5’ 

#FD0644 200 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest SalI bp from end of DNA required Thermal Incubation time without 



FastDigest SanDI 

5’...G GG W C C C...3’ 

3’...C C C W G G G...5’ 

#FD2164 20 μl 




 


Formulation 

 

 

recognition sites in 5 min at 37°C in 







Dcm: may overlap – effect not determined. 


Digestion time with 1 μl of FastDigest SanDI bp from end of DNA required Thermal Incubation time without 



FastDigest SapI 

5’...G C T C T T C(N) 1...3’ 

3’...C G A G A A G(N) 4 ...5’ 

#FD1934 20 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest SapI bp from end of DNA required Thermal Incubation time without 




62 

FastDigest Sau3AI 

5’...G A T C ...3’ 

3’... C T A G ...5’ 

#FD0784 40 μl 




Digestion time with 1 μl of FastDigest Sau3AI bp from end of DNA required Thermal Incubation time without 



5’...GG N C C...3’ 

3’...C C N G G...5’ 

#FD0194 100 μl 





 


FastDigest Sau96I 

 




Formulation 

 





Formulation 

 





20’ 




Note 

DpnI, FastDigest 

and FastDigest MboI all recognize the same 

sequence but have different methylation 

 




Note 

 



as GM2163. 

Digestion time with 1 μl of FastDigest Sau96I bp from end of DNA required Thermal Incubation time without 



FastDigest SbfI 

5’...C C T G C AG G...3’ 

3’...G G A C G T C C...5’ 

#FD1194 20 μl 




 



Formulation 

 







Digestion time with 1 μl of FastDigest SbfI bp from end of DNA required Thermal Incubation time without 



FastDigest ScaI 

5’...A G TA C T...3’ 

3’...T C A T G A...5’ 

#FD0434 100 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest ScaI bp from end of DNA required Thermal Incubation time without 



FastDigest ScrFI 

5’...C CN G G...3’ 

3’...G G N C C...5’ 

#FD1424 100 μl 




 

dcm – 


Formulation 

 


Buffer. 







Note 

 



as GM2163. 

Digestion time with 1 μl of FastDigest ScrFI bp from end of DNA required Thermal Incubation time without 



FastDigest SexAI 

5’...AC C W G G T...3’ 

3’...T G G W C C A...5’ 

#FD2114 50 μl 




 



Formulation 

 







EcoBI, EcoKI: may overlap – effect not determined. 

Digestion time with 1 μl of FastDigest SexAI bp from end of DNA required Thermal Incubation time without 




64 

FastDigest SfaNI 

5’...G C A T C(N) 5...3’ 

3’...C G T A G(N) 9 ...5’ 

#FD2124 20 μl 




Digestion time with 1 μl of FastDigest SfaNI bp from end of DNA required Thermal Incubation time without 



5’...CT R Y A G...3’ 

3’...G A Y R T C...5’ 

#FD1164 20 μl 





 


FastDigest SfcI 

 

DNA,1.0% agarose 



Formulation 

 





Formulation 

 









Note 

 

 




Digestion time with 1 μl of FastDigest SfcI bp from end of DNA required Thermal Incubation time without 



FastDigest SfiI 

5’...G G C C N N N NN G G C C...3’ 

3’...C C G G N N N N N C C G G...5’ 

#FD1824 150 μl 




pUC19-SfiI DNA, 0.7% agarose 


Formulation 

 

DNA with inserted SfiI recognition sites in 








 

20’ 

Note 

SfiI cleavage is impaired by 



as GM2163. 

SfiI at least 

two copies of its recognition sequence are 

required. The two sites can be on either the 

 

 

Digestion time with 1 μl of FastDigest SfiI bp from end of DNA required Thermal Incubation time without 


no cleavage sites 15 min 15 min 15 min 5 bp No 16 hours

FastDigest SmaI 

5’...C C CG G G...3’ 

3’...G G G C C C...5’ 

#FD0663 100 μl 

#FD0664 200 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest SmaI bp from end of DNA required Thermal Incubation time without 



FastDigest SnaBI 

5’...T A CG T A...3’ 

3’...A T G C A T...5’ 

#FD0404 50 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest SnaBI bp from end of DNA required Thermal Incubation time without 



FastDigest SpeI 

5’...AC T A G T...3’ 

3’...T G A T C A...5’ 

#FD1253 20 μl 

#FD1254 50 μl 




pUC19-BcuI DNA/Psp1406I, 1.0% agarose 


Formulation 

 

DNA with inserted BcuI recognition site/ 

Psp1406I fragments in 5 min at 37°C in 

1X FastDigest Buffer. The control DNA is. 







Digestion time with 1 μl of FastDigest SpeI bp from end of DNA required Thermal Incubation time without 

Lambda, 1μg/20μl Plasmid DNA, 1μg/20μl PCR product, ~0.2μg/30μl Genomic DNA, 1μg/10μl for complete digestion inactivation star activity 

no cleavage sites 5 min 5 min 5 min 1 bp No 16 hours 


66 

FastDigest SphI 

5’...G C A T GC...3’ 

3’...C G T A C G...5’ 

#FD0604 20 μl 




Digestion time with 1 μl of FastDigest SphI bp from end of DNA required Thermal Incubation time without 



FastDigest SspI 

5’...A A TA T T...3’ 

3’...T T A T A A...5’ 

#FD0774 100 μl 





 


 




Formulation 

 





Formulation 

 










Digestion time with 1 μl of FastDigest SspI bp from end of DNA required Thermal Incubation time without 



FastDigest StuI 

5’...A G GC C T...3’ 

3’...T C C G G A...5’ 

#FD0424 100 μl 




 



Formulation 

 










Note 

 



as GM2163. 

Digestion time with 1 μl of FastDigest StuI bp from end of DNA required Thermal Incubation time without 



FastDigest StyI 

5’...CC W W G G...3’ 

3’...G G W W C C...5’ 

#FD0414 200 μl 




 



Formulation 

 







Digestion time with 1 μl of FastDigest StyI bp from end of DNA required Thermal Incubation time without 



FastDigest SwaI 

5’...A T T TA A A T...3’ 

3’...T A A A T T T A...5’ 

#FD1244 200 μl 




M13mp18 DNA/MvaI, 1.0% agarose 


Formulation 

 

M13mp18 DNA/MvaI fragments in 5 min at 

37°C in 1X FastDigest Buffer. 







Digestion time with 1 μl of FastDigest SwaI bp from end of DNA required Thermal Incubation time without 


no cleavage sites 20 min 30 min 30 min 2 bp 65°C, 15 min 1 hour 

FastDigest TaaI 

5’...A C NG T...3’ 

3’...T G N C A...5’ 

#FD1364 20 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest TaaI bp from end of DNA required Thermal Incubation time without 




68 

FastDigest TaiI 

5’... A C G T...3’ 

3’... T G C A ...5’ 

#FD1144 50 μl 





 



Formulation 

 









Digestion time with 1 μl of FastDigest TaiI bp from end of DNA required Thermal Incubation time without 



FastDigest TaqI 

5’...TC G A...3’ 

3’...A G C T...5’ 

#FD0674 400 μl 




 

dam – 


Formulation 

 


Buffer. 







Note 

TaqI is blocked by overlapping 



Digestion time with 1 μl of FastDigest TaqI bp from end of DNA required Thermal Incubation time without 



FastDigest TatI 

5’...WG T A C W...3’ 

3’...W C A T G W...5’ 

#FD1294 20 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest TatI bp from end of DNA required Thermal Incubation time without 



FastDigest TauI 

5’...G C S GC...3’ 

3’...C G S C G...5’ 

#FD1654 20 μl 




 



Formulation 

 








Note 

 





 


Digestion time with 1 μl of FastDigest TauI bp from end of DNA required Thermal Incubation time without 



FastDigest TfiI 

5’...GA W T C...3’ 

3’...C T W A G...5’ 

#FD1784 50 μl 




 



Formulation 

 









Digestion time with 1 μl of FastDigest TfiI bp from end of DNA required Thermal Incubation time without 



FastDigest Tru1I 

5’...TT A A ...3’ 

3’...A A T T...5’ 

#FD0984 50 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest Tru1I bp from end of DNA required Thermal Incubation time without 




70 

FastDigest Tsp509I 

5’...A A T T ...3’ 

3’... T T A A ...5’ 

#FD1354 100 μl 




Digestion time with 1 μl of FastDigest Tsp509I bp from end of DNA required Thermal Incubation time without 



5’... N N C A S T G N N...3’ 

3’... N N G T S A C N N ...5’ 

#FD2104 100 μl 






FastDigest TspRI 

 




Formulation 

 





Formulation 

 











Note 

 

fragments with a 9-base 3’-extension. This 

may result in atypical DNA band patterns. 

 






 


Digestion time with 1 μl of FastDigest TspRI bp from end of DNA required Thermal Incubation time without 



FastDigest XapI 

5’...RA A T T Y...3’ 

3’...Y T T A A R...5’ 

#FD1383 50 μl 

#FD1384 100 μl 




 



Formulation 

 








Digestion time with 1 μl of FastDigest XapI bp from end of DNA required Thermal Incubation time without 



FastDigest XbaI 

5’...TC T A G A...3’ 

3’...A G A T C T...5’ 

#FD0684 300 μl 




#FD0685 750 μl 




 

dam – 


Formulation 

 

DNA dam – /SmaI fragments in 5 min at 37°C in 








Digestion time with 1 μl of FastDigest XbaI bp from end of DNA required Thermal Incubation time without 



FastDigest XhoI 

5’...CT C G A G...3’ 

3’...G A G C T C...5’ 

#FD0694 400 μl 




#FD0695 1200 μl 




 



Formulation 

 






20’ 

Note 

XbaI is blocked by overlapping 





CpG: completely overlaps – cleavage impaired 

 

Digestion time with 1 μl of FastDigest XhoI bp from end of DNA required Thermal Incubation time without 




72 

Protocols and Recommendations 

1.1. Fast DNA digestion 

1. Prepare the reaction mixture at room temperature 

in the order indicated: 

Component 

Water, 

nuclease-free* 

 

10X FastDigest 

Buffer or 

10X FastDigest 

Green Buffer 

DNA* 

Plasmid 

DNA 

up to 

20 μl 


Volume 

PCR product 

unpurified purified 

Genomic 

DNA 

17 μl up to 30 μl up to 50 μl 

2 μl 2 μl 3 μl 5 μl 

1 μg 

 

 

10 μl 

 

5 μg** 

FastDigest 

enzyme 

1 μl 1 μl 1 μl 5 μl 

Total volume 20 μl 30 μl 30 μl 50 μl 

2. Mix gently and spin down. 

3. Incubate at 37°C in a heat block or water 

thermostat for 5-15 min.*** 

4. *** 

Note 

* The volume of water should be adjusted to 

maintain the indicated total reaction volume. 

The volume of DNA can be scaled up to 

10 μl or down to 0.5 μl depending on the 

DNA concentration. 

** It is recommended that the volume of DNA 

sample should not exceed 30% of the total 

reaction volume. 

*** See the Certificate of Analysis or Table 1.3 

 

incubation time, recommended temperature 

and enzyme inactivation conditions. 

5. For fast simultaneous plasmid vector 

linearization and dephosphorylation protocol, 

see p.295. 

1.2. Reaction set-up for 

digestion of multiple DNA 

samples 

1. Pipette 2 μl of each DNA* sample into 

labeled tubes. 

2. Prepare a master mix for n+1 samples. 

 

 

Water, nuclease-free* 

10X FastDigest Buffer or 

10X FastDigest Green Buffer 

 

FastDigest enzyme 

3. Add 18 μl of master mix* into tubes 

containing DNA. 

Note 

* The volume of DNA can be scaled up to 

10 μl or down to 0.5 μl depending on the 

DNA concentration. The volume of water and 

master mix should be adjusted to maintain 

the indicated total reaction volume. 

1.3. Double and multiple 

digestion of DNA 

FastDigest enzymes allow simultaneous 

digestion of DNA with two or more enzymes in 

one digestion reaction. 

 

reaction conditions appropriately. 

 

should not exceed 1/10 of the total reaction 

volume. 

 

temperature perform sequential DNA 

cleavage: complete the first digestion 

reaction at the lower temperature, add the 

second enzyme and increase the digestion 

temperature for the second enzyme 

cleavage. 

1.4. Scaling up DNA digestion 

reaction 

DNA 1 μg 2 μg 3 μg 4 μg 5 μg 

FastDigest enzyme 

10X FastDigest Buffer 

1 μl 2 μl 3 μl 4 μl 5 μl 

or 10X FastDigest Green 

Buffer 

2 μl 2 μl 3 μl 4 μl 5 μl 

Total volume 20 μl 20 μl 30 μl 40 μl 50 μl


Table 1.3. Reaction conditions for Thermo Scientific FastDigest restriction enzymes. 

FastDigest 


Specificity 

5’3’ 

Reaction 

temp. 

Lambda 

DNA 

1μg/20μl 

Digestion time with 1 μl of 

FastDigest enzyme, min 

Plasmid 

DNA 

1μg/20μl 

PCR 

product 

~0.2μg/30μl 

Genomic 

DNA 

1μg/10μl 

bp from end of 

DNA required 

for complete 

digestion 

Thermal 

inactivation 

Incubation 

time 

without 

star 

activity 

FastDigest AatII GACGTC 37°C 15 20 15 15 5 80°C, 5 min 16 h 

FastDigest AccI GTMKAC 37°C 5 5 60 5 2 65°C, 5 min 16 h 

FastDigest Acc65I GGTACC 37°C 5 5 5 5 3 65°C, 5 min 16 h 

FastDigest AciI 37°C 5 5 5 5 2 65°C, 5 min 4 h 

FastDigest AclI AACGTT 37°C 5 5 5 5 3 65°C, 5 min 16 h 

FastDigest AcuI * 37°C 15 30 15 15 2 65°C, 5 min 16 h 

FastDigest AfeI AGCGCT 37°C 5 5 5 5 4 65°C, 5 min 16 h 

FastDigest AflII CTTAAG 37°C 5 5 5 5 4 NO 16 h 

FastDigest AgeI ACCGGT 37°C 5 5 5 5 3 80°C, 5 min 16 h 

FastDigest AjuI* 7 37°C 5 5 10 5 ND 65°C, 5 min 16 h 

FastDigest AleI CACNNNNGTG 37°C 5 5 10 5 3 65°C, 5 min 6 h 

FastDigest AluI AGCT 37°C 15 15 15 15 4 65°C, 5 min 16 h 

FastDigest Alw21I GWGCWC 37°C 5 5 5 5 1 80°C, 20 min 16 h 

FastDigest Alw26I 37°C 5 5 5 5 1 65°C, 5 min 16 h 

FastDigest AlwNI CAGNNNCTG 37°C 5 5 5 5 4 65°C, 10 min 16 h 

FastDigest ApaI GGGCCC 37°C 5 5 20 10 2 65°C, 5 min 16 h 

FastDigest ApaLI GTGCAC 37°C 5 60 10 5 4 80°C, 5 min 16h 

FastDigest AscI GGCGCGCC 37°C 5 5 5 5 2 65°C, 20 min 16 h 

FastDigest AseI ATTAAT 37°C 5 5 5 30 2 65°C, 5 min 16 h 

FastDigest AsiSI GCGATCGC 37°C no sites 5 60 5 5 80°C, 5 min 6 h 

FastDigest AvaI CYCGRG 37°C 5 5 5 5 2 65°C, 5 min 16 h 

FastDigest AvaII GGWCC 37°C 5 5 5 5 1 80°C, 20 min 16 h 

FastDigest AvrII CCTAGG 37°C 5 10 5 10 2 NO 16 h 

FastDigest BamHI GGATCC 37°C 5 5 5 5 2 80°C, 5 min 1 h 

FastDigest BanI GGYRCC 37°C 5 60 5 15 2 65°C, 10 min 16 h 

FastDigest BbsI 37°C 5 5 5 5 1 65°C, 10 min 16 h 

FastDigest BbvI 37°C 5 5 5 10 2 65°C, 5 min 0.5 h 

FastDigest BclI TGATCA 37°C 5 5 15 5 3 80°C, 20 min 16 h 

FastDigest BfaI CTAG 37°C 5 15 5 20 3 80°C, 5 min 16 h 

FastDigest BglI GCCNNNNNGGC 37°C 5 5 5 5 3 65°C, 5 min 2 h 

FastDigest BglII AGATCT 37°C 5 20 30 20 3 NO 16 h 

FastDigest BlpI GCTNAGC 37°C 5 5 5 5 2 80°C, 5 min 16 h 

FastDigest Bme1580I GKGCMC 37°C 5 5 5 30 3 80°C, 15 min 16 h 

FastDigest BmtI GCTAGC 37°C 5 5 15 5 3 80°C, 5min 1 h 

FastDigest BplI* 5 37°C 15 20 30 30 ND 65°C, 5 min 16 h 

FastDigest BpmI 30°C 15 30 15 15 2 65°C, 5 min 16 h 

FastDigest Bpu10I 37°C 15 15 30 15 3 80°C, 5 min 1 h 

FastDigest BsaAI YACGTR 37°C 5 5 5 5 1 65°C, 5 min 6 h 

FastDigest BsaBI GATNNNNATC 65°C 5 5 5 5 3 NO 1 h 

FastDigest BsaHI GRCGYC 37°C 5 5 5 5 1 65°C, 5 min 16 h 

FastDigest BsaJI CCNNGG 37°C 5 5 5 5 3 80°C, 5 min 16 h 

FastDigest BseGI 37°C 5 5 5 5 2 80°C, 5 min 16 h 

FastDigest BseNI 65°C 5 5 5 5 2 80°C, 5 min 16 h 

FastDigest BseXI 65°C 15 15 15 15 3 80°C, 15 min 16 h 

FastDigest Bsh1236I CGCG 37°C 5 5 5 5 1 80°C, 10 min 16 h 

FastDigest BsiEI CGRYCG 37°C 15 15 15 15 3 80°C, 15 min 1 h 

FastDigest BsiWI CGTACG 37°C 15 15 15 15 3 65°C, 5 min 16 h 

 


74 


FastDigest 



Specificity 

5’3’ 

Reaction 

temp. 

Lambda 

DNA 

1μg/20μl 



Plasmid 

DNA 

1μg/20μl 

PCR 

product 

~0.2μg/30μl 

Genomic 

DNA 

1μg/10μl 


DNA required 

for complete 

digestion 

Thermal 

inactivation 

Incubation 

time 

without 

star 

activity 

FastDigest BslI CCNNNNNNNGG 37°C 5 5 5 5 2 NO 16 h 

FastDigest BsmBI * 37°C 5 5 5 5 1 65°C, 10 min 6 h 

FastDigest BsmFI * 37°C 15 15 15 15 3 65°C, 5 min 16 h 

FastDigest Bsp119I TTCGAA 37°C 5 5 5 5 1 80°C, 5 min 16 h 

FastDigest Bsp120I GGGCCC 37°C 5 5 15 5 3 80°C, 10 min 16 h 

FastDigest Bsp1286I GDGCHC 37°C 5 5 5 5 2 80°C, 15 min 1 h 

FastDigest Bsp1407I TGTACA 37°C 5 5 5 5 3 80°C, 10 min 16 h 

FastDigest BspCNI * 55°C 15 30 15 15 2 80°C, 5 min 16 h 

FastDigest BspHI TCATGA 37°C 5 10 5 10 3 80°C, 5 min 0.5 h 

FastDigest BspMI * 37°C 15 15 60 20 5 65°C, 5 min 16 h 

FastDigest BsrBI 37°C 5 5 5 5 3 65°C, 5 min 16 h 

FastDigest BsrDI 55°C 15 15 15 15 3 80°C, 5 min 16 h 

FastDigest BsrFI RCCGGY 37°C 5 5 10 5 2 NO 2 h 

FastDigest BssHII GCGCGC 37°C 5 5 5 5 3 80°C, 5 min 16 h 

FastDigest BstXI CCANNNNNNTGG 37°C 5 5 5 5 4 80°C, 5 min 0.5 h 

FastDigest BstZ17I GTATAC 37°C 5 5 5 10 3 NO 6 h 

FastDigest Bsu36I CCTNAGG 37°C 5 5 5 5 2 80°C, 10 min 16 h 

FastDigest ClaI ATCGAT 37°C 5 5 5 5 2 65°C, 15 min 16 h 

FastDigest Csp6I GTAC 37°C 5 5 5 5 2 80°C, 10 min 16 h 

FastDigest DdeI CTNAG 37°C 5 5 5 10 3 65°C, 5 min 16 h 

FastDigest DpnI Gm6ATC 37°C ND 5 

not 

applicable 

10 1 80°C, 5 min 16 h 

FastDigest DraI TTTAAA 37°C 5 5 5 5 4 65°C, 5 min 16 h 

FastDigest DraIII CACNNNGTG 37°C 5 5 5 5 2 80°C, 5 min 1 h 

FastDigest DrdI GACNNNNNNGTC 37°C 5 5 5 5 1 80°C, 10 min 1 h 

FastDigest EagI CGGCCG 37°C 5 20 20 5 3 65°C, 5 min 16 h 

FastDigest Eam1105I GACNNNNNGTC 37°C 5 5 5 5 3 65°C, 5 min 1 h 

FastDigest EarI 37°C 5 5 5 5 3 80°C, 5 min 6 h 

FastDigest Ecl136II GAGCTC 37°C 5 5 5 5 1 65°C, 5 min 6 h 

FastDigest Eco31I 37°C 5 5 5 5 3 65°C, 5 min 16 h 

FastDigest Eco91I GGTNACC 37°C 5 5 5 5 2 65°C, 10 min 2 h 

FastDigest EcoNI CCTNNNNNAGG 37°C 5 5 5 20 2 65°C, 5 min 16 h 

FastDigest EcoO109I RGGNCCY 37°C 5 10 15 5 2 65°C, 5 min 16 h 

FastDigest EcoRI GAATTC 37°C 5 5 20 5 2 80°C, 5 min 0.5 h 

FastDigest EcoRV GATATC 37°C 5 5 5 5 2 NO 16 h 

FastDigest EheI GGCGCC 37°C 5 5 5 5 2 65°C, 5 min 6 h 

FastDigest Fnu4HI GCNGC 37°C 5 5 10 5 3 65°C, 5 min 16 h 

FastDigest FokI 37°C 5 5 5 5 1 65°C, 5 min 1 h 

FastDigest FspI TGCGCA 37°C 5 5 5 5 3 65°C, 15 min 16 h 

FastDigest FspAI RTGCGCAY 37°C 5 5 5 5 4 65°C, 5 min 16 h 

FastDigest HaeII RGCGCY 37°C 5 5 5 5 2 65°C, 10 min 1h 

FastDigest HaeIII GGCC 37°C 5 5 5 5 3 NO 16 h 

FastDigest HgaI 37°C 15 15 30 15 2 80°C, 5 min 1 h 

FastDigest HhaI GCGC 37°C 5 5 5 5 1 NO 16 h 

FastDigest HincII GTYRAC 37°C 5 5 5 10 1 65°C, 5 min 16 h 

FastDigest HindIII AAGCTT 37°C 5 5 20 10 3 80°C, 10 min 16 h 

FastDigest HinfI GANTC 37°C 5 5 5 5 2 65°C, 20 min 16 h 

FastDigest HinP1I GCGC 37°C 5 10 5 5 2 80°C, 10 min 16 h 

FastDigest HpaI GTTAAC 37°C 5 5 5 5 3 65°C, 20 min 0.5 h 

FastDigest HpaII CCGG 37°C 5 5 5 5 3 65°C, 5 min 16 h 

FastDigest Hpy8I GTNNAC 37°C 5 5 5 10 2 80°C, 5 min 16 h 

FastDigest HpyF10VI GCNNNNNNNGC 37°C 5 5 5 5 2 80°C, 5 min 16 h


FastDigest 


Specificity 

5’3’ 

Reaction 

temp. 

Lambda 

DNA 

1μg/20μl 



Plasmid 

DNA 

1μg/20μl 

PCR 

product 

~0.2μg/30μl 

Genomic 

DNA 

1μg/10μl 


DNA required 

for complete 

digestion 

Thermal 

inactivation 

Incubation 

time 

without 

star 

activity 

FastDigest KpnI GGTACC 37°C 5 5 5 5 3 80°C, 5 min 16 h 

FastDigest Kpn2I TCCGGA 37°C 5 5 5 5 2 80°C, 5 min 16 h 

FastDigest MauBI CGCGCGCG 37°C no sites 5 10 5 5 65°C, 5 min 16 h 

FastDigest MboI GATC 37°C 5 5 5 10 1 65°C, 15 min 16 h 

FastDigest MboII 37°C 5 5 5 5 2 65°C, 5 min 16 h 

FastDigest MfeI CAATTG 37°C 5 5 5 5 3 NO 16 h 

FastDigest MluI ACGCGT 37°C 5 5 5 15 3 80°C, 5 min 16 h 

FastDigest MlyI 37°C 5 5 30 5 2 80°C, 5 min 1 h 

FastDigest MnlI 37°C 5 5 5 5 2 65°C, 5 min 16 h 

FastDigest MreI CGCCGGCG 37°C no sites 5 5 5 3 80°C, 5 min 16 h 

FastDigest MscI TGGCCA 37°C 5 5 30 20 3 80°C, 20 min 16 h 

FastDigest MseI TTAA 37°C 5 5 5 5 4 65°C, 5 min 16 h 

FastDigest MslI CAYNNNNRTG 37°C 5 5 5 5 4 65°C, 20 min 16 h 

FastDigest MspI CCGG 37°C 5 5 5 5 3 NO 16 h 

FastDigest MssI GTTTAAAC 37°C 5 5 5 5 2 65°C, 10 min 16 h 

FastDigest MvaI CCWGG 37°C 5 5 5 10 4 NO 1 h 

FastDigest Mva1269I 37°C 5 5 5 5 3 65°C, 5 min 16 h 

FastDigest NaeI GCCGGC 37°C ND 5 5 5 3 65°C, 15 min 16 h 

FastDigest NciI CCSGG 37°C 5 5 5 5 3 80°C, 20 min 16 h 

FastDigest NcoI CCATGG 37°C 5 10 10 5 3 65°C, 15 min 16 h 

FastDigest NdeI CATATG 37°C 5 5 60 30 3 65°C, 5 min 6 h 

FastDigest NheI GCTAGC 37°C 5 15 5 5 5 65°C, 5 min 6 h 

FastDigest NlaIII CATG 37°C 5 10 5 10 4 80°C, 5 min 16 h 

FastDigest NlaIV GGNNCC 37°C 5 5 5 5 2 65°C, 20 min 16 h 

FastDigest NmuCI GTSAC 37°C 5 5 5 10 2 65°C, 5 min 16 h 

FastDigest NotI GCGGCCGC 37°C no sites 30 5 10 2 80°C, 5 min 16 h 

FastDigest NruI TCGCGA 37°C 5 5 5 5 5 NO 4 h 

FastDigest NsiI ATGCAT 37°C 5 5 5 10 3 65°C, 15 min 6 h 

FastDigest NspI RCATGY 37°C 5 5 5 5 2 65°C, 5 min 16 h 

FastDigest PacI TTAATTAA 37°C no sites 5 5 5 2 65°C, 10 min 16 h 

FastDigest PdmI GAANNNNTTC 37°C 5 5 5 5 2 65°C, 5 min 16 h 

FastDigest PflMI CCANNNNNTGG 37°C 5 5 5 5 3 65°C, 10 min 6 h 

FastDigest PfoI TCCNGGA 37°C 5 5 5 5 3 65°C, 5 min 16 h 

FastDigest PmlI CACGTG 37°C 5 5 5 5 2 80°C, 10 min 1 h 

FastDigest PpuMI RGGWCCY 37°C 5 5 5 20 2 80°C, 5 min 1 h 

FastDigest PshAI GACNNNNGTC 37°C 5 5 5 5 3 80°C, 20 min 16 h 

FastDigest PsiI TTATAA 37°C 5 5 5 5 5 65°C, 5 min 16 h 

FastDigest PspFI 37°C 5 5 5 5 4 80°C, 5 min 16 h 

FastDigest PstI CTGCAG 37°C 5 5 30 5 3 NO 16 h 

FastDigest PsuI RGATCY 37°C 5 5 5 5 2 80°C, 5 min 16 h 

FastDigest PsyI GACNNNGTC 37°C 5 5 5 5 2 80°C, 5 min 4 h 

FastDigest PvuI CGATCG 37°C 5 15 5 5 4 80°C, 5 min 16 h 

FastDigest PvuII CAGCTG 37°C 5 5 5 5 1 NO 0.5 h 

FastDigest RsaI GTAC 37°C 5 5 5 5 3 NO 16 h 

FastDigest RsrII CGGWCCG 37°C 5 5 5 5 1 65°C, 5 min 16 h 

FastDigest SacI GAGCTC 37°C 5 15 30 10 1 65°C, 5 min 16 h 

FastDigest SalI GTCGAC 37°C 5 5 60 5 3 65°C, 10 min 16 h 

FastDigest SanDI GGGWCCC 37°C 5 5 5 10 5 NO 16 h 

FastDigest SapI 37°C 5 5 5 5 2 65°C, 5 min 16 h 

FastDigest Sau3AI GATC 37°C 5 5 10 5 4 65°C, 20 min 16 h 

FastDigest Sau96I GGNCC 37°C 5 5 5 5 2 NO 16 h 

FastDigest SbfI CCTGCAGG 37°C 5 5 5 5 3 NO 1 h 

 


76 


FastDigest 


Note 

* – for digestion with FastDigest 

for digestion with FastDigest AjuI and FastDigest 

for digestion with FastDigest BplI and FastDigest 

for digestion with FastDigest 

NO – no thermal inactivation. Spin column purification or phenol/chloroform extraction and ethanol precipitation of 

DNA is recommended. 

ND – not determined. 


Specificity 

5’3’ 

Reaction 

temp. 

Lambda 

DNA 

1μg/20μl 



Plasmid 

DNA 

1μg/20μl 

PCR 

product 

~0.2μg/30μl 

Genomic 

DNA 

1μg/10μl 


DNA required 

for complete 

digestion 

Thermal 

inactivation 

Incubation 

time 

without 

star 

activity 

FastDigest ScaI AGTACT 37°C 5 5 5 5 4 65°C, 10 min 16 h 

FastDigest ScrFI CCNGG 37°C 5 5 5 5 4 NO 16 h 

FastDigest SexAI ACCWGGT 37°C 5 10 5 15 4 65°C, 5 min 16 h 

FastDigest SfaNI 37°C 5 5 5 5 1 65°C, 5 min 16 h 

FastDigest SfcI CTRYAG 37°C 5 5 5 5 2 65°C, 20 min 16 h 

FastDigest SfiI GGCCNNNNNGGCC 50°C no sites 15 15 15 5 NO 16 h 

FastDigest SmaI CCCGGG 37°C 5 5 5 5 1 65°C, 5 min 16 h 

FastDigest SnaBI TACGTA 37°C 5 5 5 5 3 65°C, 5 min 16 h 

FastDigest SpeI ACTAGT 37°C no sites 5 5 5 1 NO 16 h 

FastDigest SphI GCATGC 37°C 5 5 5 5 5 65°C, 5 min 16 h 

FastDigest SspI AATATT 37°C 5 5 5 30 2 65°C, 5 min 1 h 

FastDigest StuI AGGCCT 37°C 5 5 5 5 3 80°C, 10 min 16 h 

FastDigest StyI CCWWGG 37°C 5 5 20 20 3 65°C, 5 min 16 h 

FastDigest SwaI ATTTAAAT 37°C no sites 20 30 30 2 65°C, 15 min 1 h 

FastDigest TaaI ACNGT 65°C 5 5 5 5 3 NO 16 h 

FastDigest TaiI ACGT 65°C 5 5 5 5 2 NO 6 h 

FastDigest TaqI TCGA 65°C 5 5 5 5 4 NO 16 h 

FastDigest TatI WGTACW 65°C 15 15 15 20 5 NO 1 h 

FastDigest TauI GCSGC 55°C 15 60 15 30 5 NO 16 h 

FastDigest TfiI GAWTC 37°C 5 5 10 5 2 65°C, 5 min 16 h 

FastDigest Tru1I TTAA 65°C 5 5 5 10 3 NO 2 h 

FastDigest Tsp509I AATT 65°C 5 5 5 5 3 NO 6 h 

FastDigest TspRI 65°C 5 5 5 5 2 NO 6 h 

FastDigest XapI RAATTY 37°C 5 5 5 10 4 80°C, 5 min 6 h 

FastDigest XbaI TCTAGA 37°C 5 5 5 10 2 65°C, 20 min 16 h 

FastDigest XhoI CTCGAG 37°C 5 5 5 10 2 80°C, 5 min 16 h 







S = C or G;


Thermo Scientific conventional restriction 

endonucleases are a large collection of high 

quality restriction enzymes. The enzymes are 

optimized to work in one of the buffers in our 

Five Buffer System. In addition, a universal 

Tango buffer is provided for convenience in 

double digestions. All of the enzymes exhibit 

Activity and Quality 

Control Assays 

Activity Assay 

One unit is the amount of enzyme required 

to hydrolyze 1 μg of substrate DNA in 

60 min in 50 μl of reaction mixture under 

the recommended conditions. To determine 

restriction enzyme activity, concentrated 

enzymes are first diluted to approximately 

0.5-1 units/μl with an enzyme dilution 

 

 

2-mercaptoethanol, 10% glycerol and 0.2 mg/ 

 

In general, enzymes are assayed with phage 

DNA at 37°C. However, some exceptions apply: 

 

activity at temperatures other than 37°C are 

assayed under their optimal temperature. 

 

on DNA are assayed with another specific 

DNA substrate. 

 

sites on the DNA are assayed using DNA 

hydrolyzed with another restriction enzyme. 

 

methylation are assayed with DNA 

dam – , dcm – 



The labelled oligonucleotides are incubated 

with an excess of restriction enzyme, separated 

on a polyacrylamide gel under denaturing 

conditions and analyzed by phospho-imaging. 

The restriction enzyme passes this quality 

control test if there is no degradation of labelled 

oligonucleotides and no decrease in specific 

see 

100% activity in the recommended buffer 

and reaction conditions. To ensure consistent 

enzyme performance, Thermo Scientific 

restriction enzyme reaction buffers contain 

premixed BSA, which enhances the stability of 

many enzymes and binds contaminants that 

may be present in DNA preparations. 

Non-specific Nuclease 

and Cross-contamination Assay 

Varying amounts of restriction enzyme 

 

1 μg of substrate DNA under the recommended 

assay conditions. After electrophoretic 

separation of the DNA fragments, the 

characteristic banding patterns are examined 

for alterations. 

To pass the test, the restriction enzyme must 

yield an unaltered banding pattern under 

 

 

restriction enzyme star activity, see the product 

description or Certificate of Analysis supplied 

with each enzyme. 

Blue/White (B/W) Cloning Assay 

The B/W assay was replaced by LO test after 

validating experiments showed LO test ability to 

detect nuclease and phosphatase activities with 

sensitivity that equals to that of B/W test. 

Ligation and Recleavage (L/R) Assay 

The ligation and recleavage assay was replaced 

with LO test after validating experiments 

revealed LO test ability to trace nuclease and 

phosphatase activities with sensitivity that is 

higher than L/R test by a factor of 100. 


All conventional restriction enzymes should 

be stored at -20°C. During shipment on dry ice, 

enzymes may freeze. This does not affect their 

quality – all Thermo Scientific enzymes 

are 100% active after at least three 

freeze-thaw cycles. 


shipped on blue ice since their quality is not 

affected by short exposure to 4°C. 


78 

Product List and Cross-reference to FastDigest Restriction Enzymes 

Table 1.4. Thermo Scientific conventional restriction enzymes. 

Conventional 


FastDigest 



AanI FastDigest PsiI PsiI TTATAA ER2061 82 

AarI AarI ER1581/2 82 

AatII FastDigest AatII AatII GACGTC ER0991/2 82 

Acc65I FastDigest Acc65I GGTACC ER0901/2 83 

AdeI FastDigest DraIII DraIII CACNNNGTG ER1231 83 

AjiI BtrI CACGTC ER1941 84 

AjuI FastDigest AjuI AjuI 7 ER1951 84 

AlfI AlfI 6 ER1801 85 

AloI AloI 6 ER1491 85 

AluI FastDigest AluI AluI AGCT ER0011/2 85 

Alw21I FastDigest Alw21I HgiAI GWGCWC ER0021 86 

Alw26I FastDigest Alw26I BsmAI ER0031 86 

Alw44I FastDigest ApaLI ApaLI GTGCAC ER0041 86 

ApaI FastDigest ApaI ApaI GGGCCC ER1411/5 87 

BamHI FastDigest BamHI BamHI GGATCC ER0051/2/3/5 88 

BauI BsiI ER1841 88 

BclI FastDigest BclI BclI TGATCA ER0721/2/5 89 

BcnI FastDigest NciI CauII CCSGG ER0061 89 

BcuI FastDigest SpeI SpeI ACTAGT ER1251/2 89 

BfmI FastDigest SfcI SfeI CTRYAG ER1161/2 90 

BfuI BciVI ER1501 90 

BglI FastDigest BglI BglI GCCNNNNNGGC ER0071/2 90 

BglII FastDigest BglII BglII AGATCT ER0081/2 91 

Bme1390I FastDigest ScrFI ScrFI CCNGG ER1421/2 91 

BoxI FastDigest PshAI PshAI GACNNNNGTC ER1431 92 

BpiI FastDigest BbsI BbvII ER1011/2 92 

BplI FastDigest BpII BplI 5 ER1311/2 92 

Bpu10I FastDigest Bpu10l Bpu10I ER1181 93 

Bpu1102I FastDigest BlpI EspI GCTNAGC ER0091/2 93 

BseDI FastDigest BsaJI SecI CCNNGG ER1081/2 94 

BseGI FastDigest BseGI ER0871/2 94 

BseJI FastDigest BsaBI BsaBI GATNNNNATC ER1711 94 

BseLI FastDigest BslI BsiYI CCNNNNNNNGG ER1201 95 

BseMI FastDigest BsrDI BsrDI ER1261/2 95 

BseMII FastDigest BspCNI BseMII ER1401 95 

BseNI FastDigest BseNI BsrI ER0881/2 96 

BseSI FastDigest Bme1580I BseSI GKGCMC ER1441 96 

BseXI FastDigest BseXI BbvI ER1451/2 96 

Bsh1236I FastDigest Bsh1236I FnuDII CGCG ER0921/2 97 

Bsh1285I FastDigest BsiEI McrI CGRYCG ER0891 97 

BshNI FastDigest BanI HgiCI GGYRCC ER1001 97 

BshTI FastDigest AgeI AgeI ACCGGT ER1461/2 98 

Bsp68I FastDigest NruI NruI TCGCGA ER0111 98 

Bsp119I FastDigest Bsp119I AsuII TTCGAA ER0121 99 

Bsp120I FastDigest Bsp120I GGGCCC ER0131 99 

Bsp143I FastDigest Sau3AI MboI GATC ER0781/2 99 

Bsp1407I FastDigest Bsp1407I Bsp1407I TGTACA ER0931/2 100 

BspLI FastDigest NlaIV NlaIV GGNNCC ER1151/2 100 

BspOI FastDigest BmtI GCTAGC ER2041 100 

BspPI BinI ER1321/2 101 

BspTI FastDigest AflII AflII CTTAAG ER0831 101 



Conventional 


FastDigest 



Bst1107I FastDigest BstZ17I SnaI GTATAC ER0701 102 

BstXI FastDigest BstXI BstXI CCANNNNNNTGG ER1021/2 102 

Bsu15I FastDigest ClaI ClaI ATCGAT ER0141/2/5 103 

BsuRI FastDigest HaeIII HaeIII GGCC ER0151 103 

BveI FastDigest BspMI BspMI ER1741 104 

CaiI FastDigest AlwNI AlwNI CAGNNNCTG ER1391 104 

Cfr9I CCCGGG ER0171/2 105 

Cfr10I FastDigest BsrFI Cfr10I RCCGGY ER0181 105 

Cfr13I FastDigest Sau96I AsuI GGNCC ER0191 105 

Cfr42I SacII CCGCGG ER0201/2/5 106 

CpoI FastDigest RsrII RsrII CGGWCCG ER0741/2 106 

CseI FastDigest HgaI HgaI ER1901 106 

Csp6I FastDigest Csp6I GTAC ER0211 107 

DpnI FastDigest DpnI DpnI Gm6ATC ER1701/2/5 107 

DraI FastDigest DraI AhaIII TTTAAA ER0221/3 108 

Eam1104I FastDigest EarI Ksp632I ER0231/2 108 

Eam1105I FastDigest Eam1105I Eam1105I GACNNNNNGTC ER0241 108 

Ecl136II FastDigest Ecl136II GAGCTC ER0251 109 

Eco24I GRGCYC ER0281 109 

Eco31I FastDigest Eco31I Eco31I ER0291/2 110 

Eco32I FastDigest EcoRV EcoRV GATATC ER0301/2/3/5 110 

Eco47I FastDigest AvaII AvaII GGWCC ER0311/2 110 

Eco47III FastDigest AfeI Eco47III AGCGCT ER0321/2 111 

Eco52I FastDigest EagI XmaIII CGGCCG ER0331/2 111 

Eco57I FastDigest AcuI Eco57I ER0341/2 112 

Eco72I FastDigest PmlI PmaCI CACGTG ER0361 112 

Eco81I FastDigest Bsu36I SauI CCTNAGG ER0371/2 113 

Eco88I FastDigest AvaI AvaI CYCGRG ER0381 113 

Eco91I FastDigest Eco91I BstEII GGTNACC ER0391/2 113 

Eco105I FastDigest SnaBI SnaBI TACGTA ER0401/2 114 

Eco130I FastDigest StyI StyI CCWWGG ER0411 114 

Eco147I FastDigest StuI StuI AGGCCT ER0421/2 114 

EcoO109I FastDigest EcoO109I DraII RGGNCCY ER0261 115 

EcoRI FastDigest EcoRI EcoRI GAATTC ER0271/2/3/5 115 

EcoRII EcoRII CCWGG ER1921 116 

EheI FastDigest EheI GGCGCC ER0441 116 

Esp3I FastDigest BsmBI Esp3I ER0451/2 117 

FaqI FastDigest BsmFI FinI ER1811 117 

FspAI FastDigest FspAI FspAI RTGCGCAY ER1661/2 118 

FspBI FastDigest BfaI MaeI CTAG ER1761/2 118 

GsuI FastDigest BpmI GsuI ER0461/2 118 

HhaI FastDigest HhaI HhaI GCGC ER1851 119 

Hin1I FastDigest BsaHI AcyI GRCGYC ER0471 119 

Hin1II FastDigest NlaIII NlaIII CATG ER1831 120 

Hin6I FastDigest HinP1I GCGC ER0481 120 

HincII FastDigest HincII HindII GTYRAC ER0491/2 120 

HindIII FastDigest HindIII HindIII AAGCTT ER0501/2/3/5 121 

HinfI FastDigest HinfI HinfI GANTC ER0801/2/3 121 

HpaII FastDigest HpaII HpaII CCGG ER0511/2 121 

HphI HphI ER1101/2 122 

Hpy8I FastDigest Hpy8I MjaIV GTNNAC ER1571/2 122 

HpyF3I FastDigest DdeI DdeI CTNAG ER1881/2 122 

HpyF10VI FastDigest HpyF10VI MwoI GCNNNNNNNGC ER1731/2 123 

 


80 


Conventional 


FastDigest 



KpnI FastDigest KpnI KpnI GGTACC ER0521/2/3 123 

Kpn2I FastDigest Kpn2I BspMII TCCGGA ER0531/2 123 

KspAI FastDigest HpaI HpaI GTTAAC ER1031/2 124 

LguI FastDigest SapI SapI ER1931/2 124 

Lsp1109I FastDigest BbvI BbvI ER2071 124 

LweI FastDigest SfaNI SfaNI ER1621/2 125 

MauBI FastDigest MauBI MauBI CGCGCGCG ER2081 125 

MbiI FastDigest BsrBI BsrBI ER1271 125 

MboI FastDigest MboI MboI GATC ER0811/2 126 

MboII FastDigest MboII MboII ER0821/2 126 

MlsI FastDigest MscI BalI TGGCCA ER1211/2 127 

MluI FastDigest MluI MluI ACGCGT ER0561/2 127 

MnlI FastDigest MnlI MnlI ER1071/2 127 

Mph1103I FastDigest NsiI AvaIII ATGCAT ER0731/2 128 

MreI FastDigest MreI Sse232I CGCCGGCG ER2021 128 

MspI FastDigest MspI HpaII CCGG ER0541/2 128 

MssI FastDigest MssI PmeI GTTTAAAC ER1341/2 129 

MunI FastDigest MfeI MfeI CAATTG ER0751/2 129 

MvaI FastDigest MvaI CCWGG ER0551 129 

Mva1269I FastDigest Mva1269I BsmI ER0961/2 130 

NcoI FastDigest NcoI NcoI CCATGG ER0571/2/5 130 

NdeI FastDigest NdeI NdeI CATATG ER0581/2/5 131 

NheI FastDigest NheI NheI GCTAGC ER0971/2/5 131 

NmuCI FastDigest NmuCI Tsp45I GTSAC ER1511 132 

NotI FastDigest NotI NotI GCGGCCGC ER0591/2/3/5 132 

NsbI FastDigest FspI MstI TGCGCA ER1221 132 

OliI FastDigest AleI OliI CACNNNNGTG ER1631/2 133 

PacI FastDigest PacI PacI TTAATTAA ER2201 133 

PaeI FastDigest SphI SphI GCATGC ER0601/2 133 

PagI FastDigest BspHI BspHI TCATGA ER1281/2 134 

PasI PasI CCCWGGG ER1861 134 

PauI FastDigest BssHII BsePI GCGCGC ER1091/2 134 

PdiI FastDigest NaeI NaeI GCCGGC ER1521/2 135 

PdmI FastDigest PdmI XmnI GAANNNNTTC ER1531/2 135 

PfeI FastDigest TfiI TfiI GAWTC ER1781 135 

Pfl23II FastDigest BsiWI SplI CGTACG ER0851 136 

PfoI FastDigest PfoI PfoI TCCNGGA ER1751 136 

Ppu21I FastDigest BsaAI BsaAI YACGTR ER1971 137 

PscI BspLU11I ACATGT ER1871/2 137 

Psp5II FastDigest PpuMI PpuMI RGGWCCY ER0761 138 

Psp1406I FastDigest AclI AclI AACGTT ER0941/2 138 

PstI FastDigest PstI PstI CTGCAG ER0611/2/5 138 

PsuI FastDigest PsuI XhoII RGATCY ER1551 139 

PsyI FastDigest PsyI Tth111I GACNNNGTC ER1331 139 

PvuI FastDigest PvuI PvuI CGATCG ER0621/2 139 

PvuII FastDigest PvuII PvuII CAGCTG ER0631/3/5 140 

RsaI FastDigest RsaI RsaI GTAC ER1121/2 140 

RseI FastDigest MslI MslI CAYNNNNRTG ER2001/2 140 

SacI FastDigest SacI SacI GAGCTC ER1131/2/5 141 

SalI FastDigest SalI SalI GTCGAC ER0641/2/5 141 

SatI FastDigest Fnu4HI Fnu4HI GCNGC ER1641/2 142 

ScaI FastDigest ScaI ScaI AGTACT ER0431/2 142 

SchI FastDigest MlyI ER1371 143 



Conventional 


FastDigest 



SdaI FastDigest SbfI Sse8387I CCTGCAGG ER1191/2 143 

SduI FastDigest Bsp1286I SduI GDGCHC ER0651 143 

SfaAI FastDigest AsiSI SgfI GCGATCGC ER2091 144 

SfiI FastDigest SfiI SfiI GGCCNNNNNGGCC ER1821 144 

SgeI SgeI ER2211 145 

SgrDI SgrDI CGTCGACG ER2031 145 

SgsI FastDigest AscI AscI GGCGCGCC ER1891/2 145 

SmaI FastDigest SmaI SmaI CCCGGG ER0661/2/3/5 146 

SmiI FastDigest SwaI SwaI ATTTAAAT ER1241 146 

SmoI SmlI CTYRAG ER1981 147 

SsiI FastDigest AciI AciI ER1791 147 

SspI FastDigest SspI SspI AATATT ER0771/2 148 

SspDI GGCGCC ER2191 148 

TaaI FastDigest TaaI Tsp4CI ACNGT ER1361/2 149 

TaiI FastDigest TaiI ACGT ER1141/2 149 

TaqI FastDigest TaqI TaqI TCGA ER0671/2 149 

TasI FastDigest Tsp509I TspEI AATT ER1351/2 150 

TatI FastDigest TatI TatI WGTACW ER1291 150 

TauI FastDigest TauI TauI GCSGC ER1651/2 150 

Tru1I FastDigest Tru1I MseI TTAA ER0981/2/3 151 

TscAI FastDigest TspRI TspRI ER2101 151 

Van91I FastDigest PflMI PflMI CCANNNNNTGG ER0711/2 152 

VspI FastDigest AseI VspI ATTAAT ER0911/2 152 

XagI FastDigest EcoNI EcoNI CCTNNNNNAGG ER1301 152 

XapI FastDigest XapI ApoI RAATTY ER1381 153 

XbaI FastDigest XbaI XbaI TCTAGA ER0681/2/3/5 153 

XceI FastDigest NspI NspI RCATGY ER1471/2 153 

XhoI FastDigest XhoI XhoI CTCGAG ER0691/2/3/5 154 

XmaJI FastDigest AvrII AvrII CCTAGG ER1561/2 154 

XmiI 

Nicking enzymes 

FastDigest AccI AccI GTMKAC ER1481/2 154 

Nb.Bpu10I Nb.Bpu10I 

CCTNA GC 

GGANTCG 

ER1681 155 

Nt.Bpu10I Nt.Bpu10I 

CCTNAGC 

GG ANTCG 

ER2011 156 

Nb.Mva1269I 

Homing enzyme 

Nb.BsmI 

GAATG C 

CTTACG 

ER2051 156 

I-SceI I-SceI 

TAGGG ATAACAGGGTAAT 

ATCCCTATT GTCCCATTA 

ER1771 157 

Alphabetic list of commercially available restriction enzymes see p.205. 


82 

Product Description 

AanI 

5’...T T AT A A ...3’ 

3’...A A T A T T ...5’ 

#ER2061 200 u 


10X Buffer Tango 1 ml 

Also available as 

FastDigest PsiI 

AarI 

5’...C A C C T G C (N) 4 ...3’ 

3’...G T G G A C G (N) 8 ...5’ 

#ER1581 


25 u 

10X Buffer AarI 1 ml 


50X oligonucleotide 25μl 

#ER1582 


125 u 

10X Buffer AarI 1 ml 


50X oligonucleotide 2x25 μl 

Note 

 

its recognition sequence are required. 

 

AarI recognition sequence in the reaction 

mixture significantly improves cleavage of 


Concentration 

10 u/μl 

20’ 

Conditions for 100% Activity 

1X Buffer Tango at 37°C. 

Storage Buffer 

AanI is supplied in: 

 

1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA and 

 

Concentration 

2 u/μl 


1X Buffer AarI at 37°C. 



AarI is supplied in: 

 

100 mM KCl, 1 mM EDTA, 1 mM DTT, 

 

DNAs, especially of those with a single AarI 

site. Still, a complete cleavage of some 

substrates with AarI is difficult to achieve. 

 

may result in star activity. 

 

AasI Thermo Scientific enzyme FastDigest DrdI 

AatII 

5’...G A C G TC...3’ 

3’...C T G C A G...5’ 

#ER0991 300 u 

#ER0992 1500 u 




FastDigest AatII 

20’ 

Concentration 

10 u/μl 




AatII is supplied in: 

 

1 mM DTT, 1 mM EDTA, 0.2 mg/ml BSA and 

 

20’ 

Activity in Five Buffer System, % 

B G O R Tango 2X Tango 

50-100 50-100 0-20 0-20 100 20-50 




Digestion of Agarose-embedded DNA 

Minimum 5 units of the enzyme are required 

for complete digestion of 1 μg of agaroseembedded 

DNA in 16 hours. 



NR NR 0-20 0-20 NR 50-100 



CpG: may overlap – cleavage impaired 

 



Minimum 5 units of the enzyme are required for 

digestion of 1 μg of agarose-embedded DNA 

in 16 hours. 

DNA. This may cause DNA band shifting 

during electrophoresis. To avoid atypical 

DNA band patterns, use the 6X DNA Loading 

 

preparation or heat the digested DNA in the 

presence of SDS prior to electrophoresis. 



50-100 20-50 0-20 0-20 100 20-50 






for complete digestion of 1μg of agaroseembedded 

DNA in 16 hours.

AccI Thermo Scientific enzymes FastDigest AccI and XmiI 

AccII Thermo Scientific enzymes FastDigest Bsh1236I and Bsh1236I 

AccIII Thermo Scientific enzymes FastDigest Kpn2I and Kpn2I 

Acc65I 

5’...GG T A C C...3’ 

3’...C C A T G G...5’ 

#ER0901 


1000 u 

10X Buffer O 1 ml 


#ER0902 


5000 u 

10X Buffer O 2x1 ml 



FastDigest Acc65I 

Concentration 

10 u/μl 


1X Buffer O at 37°C. 


Acc65I is supplied in: 

 


 

AccB7I Thermo Scientific enzymes FastDigest PflMI and Van91I 

AciI Thermo Scientific enzymes FastDigest AciI and SsiI 

AclI Thermo Scientific enzymes FastDigest AcII and Psp1406I 

AcsI Thermo Scientific enzymes FastDigest XapI and XapI 

AcuI Thermo Scientific enzymes FastDigest AcuI and Eco57I 

AcyI Thermo Scientific enzymes FastDigest BsaHI and Hin1I 

AdeI 

5’...C A C N N NG T G...3’ 

3’...G T G N N N C A C...5’ 

#ER1231 500 u 


10X Buffer G 1 ml 



FastDigest DraIII 

Concentration 

10 u/μl 

20’ 


1X Buffer G at 37°C. 


AdeI is supplied in: 

 


 



0-20 20-50 100 20-50 20-50 50-100 




 





Note 

 

dcm methylation. To avoid dcm methylation, 




0-20 100 20-50 100 100* 20-50 

* 




 

EcoKI, EcoBI may overlap – effect not determined. 





Note 

 



84 

AfaI Thermo Scientific enzymes FastDigest Csp6I and Csp6I 

FastDigest RsaI and RsaI 

AfeI Thermo Scientific enzymes FastDigest AfeI and Eco47III 

AflII Thermo Scientific enzymes FastDigest AflII and BspTI 

AgeI Thermo Scientific enzymes FastDigest AgeI and BshTI 

AhaIII Thermo Scientific enzymes FastDigest DraI and DraI 

AhdI Thermo Scientific enzymes FastDigest Eam1105I and Eam1105I 

AjiI 

5’...C A CG T C...3’ 

3’...G T G C A G...5’ 

#ER1941 200 u 


10X Buffer AjiI 1 ml 


AjuI 

5’... 7(N)GAA(N) 7TTGG(N) 11 ...3’ 

3’... 12(N)CTT(N) 7AACC(N) 6 ...5’ 

#ER1951 100 u 


10X Buffer R 1 ml 

50X SAM 0.1 ml 

10X BufferTango 1 ml 


FastDigest AjuI 


20’ Activity in Five Buffer System, % 


NR NR 20-50* NR NR 20-50* 

* 

Concentration 

5 u/μl 


1X Buffer AjiI at 37°C. 


AjiI is supplied in: 

 


 

20’ 

Concentration 

5 u/μl 


1X Buffer R at 37°C. 

SAM 0.01 mM. 


AjuI is supplied in: 

 


 












B +SAM G +SAM O +SAM R +SAM Tango +SAM 2X Tango +SAM 

0-20 50-100 20-50 100 50-100 50-100 





Note 

 

 

AjuI is difficult to achieve. 

 

maximum cleavage level achieved when 

no change in the fragmentation pattern is 

observed with further enzyme increase. 

 

sides of the interrupted recognition site. In 

certain sequence contexts, the cleavage 

position may be shifted by one base pair. 

However, the cleavage position indicated 

above will predominate.

AleI Thermo Scientific enzymes FastDigest AleI and OliI 

AlfI 

5’...10(N)GCA(N) 6TGC(N) 12 ...3’ 

3’... 12(N)CGT(N) 6ACG(N) 10 ...5’ 

#ER1801 50 u 




10X BufferTango 1 ml 

AloI 

5’... 7(N)GAAC(N) 6TCC(N) 12-13...3’ 

3’... 12-13(N)CTTG(N) 6AGG(N) 7 ...5’ 

#ER1491 100 u 




Note 

 

 

sides from the interrupted recognition site. 

Its unique feature is a degenerate cleavage 

point on the 3’ side of the recognition 

 

AluI 

5’...A GC T...3’ 

3’...T C G A...5’ 

#ER0011 


600 u 


#ER0012 


3000 u 

10X Buffer Tango 2x1 ml 


FastDigest AluI 

20’ 

Concentration 

2 u/μl 



SAM 0.01 mM. 


AlfI is supplied in: 

 


 

20’ 

Concentration 

2 u/μl 




AloI is supplied in: 

 


 

 

the reaction mixture results in incomplete 

cleavage with AloI. 

 


 


20’ 

Concentration 

10 u/μl 





0-20 0-20 0-20 100 0-20 20-50 







Note 

 

 

AlfI is difficult to achieve. 

 






0-20 0-20 0-20 100 20-50 100 


Dam: may overlap – effect not determined. 



 



 





50-100 0-20 0-20 0-20 100 20-50 


AluI is supplied in: 

 

1 mM DTT, 0.1 mM EDTA, 0.15% Triton X-100, 

 





86 

AlwI Thermo Scientific enzyme BspPI 

Alw21I 

5’...G W G C WC...3’ 

3’...C W C G W G...5’ 

#ER0021 500 u 






Alw26I 

5’...G T C T C(N) 1...3’ 

3’...C A G A G(N) 5 ...5’ 

#ER0031 1000 u 





Alw44I 

5’...GT G C A C...3’ 

3’...C A C G T G...5’ 

#ER0041 1000 u 




FastDigest ApaLI 


Concentration 

10 u/μl 



20’ 

Concentration 

10 u/μl 



Concentration 

10 u/μl 

20’ 




Alw44I is supplied in: 

 


 

AlwNI Thermo Scientific enzymes FastDigest AIwNI and CaiI 

Aor13HI Thermo Scientific enzymes FastDigest Kpn2I and Kpn2I 

Aor51HI Thermo Scientific enzymes FastDigest AfeI and Eco47III 

20’ 



0-20 20-50 100 50-100 20-50 50-100 



 


 






50-100 100 0-20 0-20 100 100 



 


 




 



50-100 100 0-20 50-100 100 50-100 







DNA in 16 hours.

ApaI 

5’...G G G C CC...3’ 

3’...C C C G G G...5’ 

#ER1411 3000 u 

#ER1415 5000 u 


10X Buffer B 2x1 ml 



FastDigest ApaI 

Concentration 

10 u/μl 


1X Buffer B at 37°C. 


ApaI is supplied in: 

 


 

20’ 



100 20-50 0-20 0-20 20-50 0-20 




 





Note 

 



ApaLI Thermo Scientific enzymes FastDigest ApaLI and Alw44I 

ApoI Thermo Scientific enzymes FastDigest XapI and XapI 

AscI Thermo Scientific enzymes FastDigest AscI and SgsI 

AseI Thermo Scientific enzymes FastDigest AseI and VspI 

AsiSI Thermo Scientific enzymes FastDigest AsiSI and SfaAI 

AspI Thermo Scientific enzymes FastDigest PsyI and PsyI 

Asp700I Thermo Scientific enzymes FastDigest PdmI and PdmI 

Asp718I Thermo Scientific enzymes FastDigest Acc65I and Acc65I ; 

FastDigest KpnI and KpnI 

AspEI Thermo Scientific enzymes FastDigest Eam1105I and Eam1105I 

AspHI Thermo Scientific enzymes FastDigest Alw21I and Alw21I 

AsuI Thermo Scientific enzymes FastDigest Sau96I and Cfr13I 

AsuII Thermo Scientific enzymes FastDigest Bsp119I and Bsp119I 

AvaI Thermo Scientific enzymes FastDigest AvaI and Eco88I 

AvaII Thermo Scientific enzymes FastDigest AvaII and Eco47I 

AvaIII Thermo Scientific enzymes FastDigest NsiI and Mph1103I 

AviII Thermo Scientific enzymes FastDigest FspI and NsbI 

AvrII Thermo Scientific enzymes FastDigest AvrII and XmaJI 

BaeGI Thermo Scientific enzymes FastDigest Bme1580I and BseSI 

BalI Thermo Scientific enzymes FastDigest MscI and MlsI 


88 

BamHI 

5’...GG A T C C...3’ 

3’...C C T A G G...5’ 

#ER0051 


4000 u 

10X Buffer BamHI 2x1 ml 


#ER0055 


10,000 u 



#ER0052 5x4000 u 

#ER0053 HC, 20,000 u 





FastDigest BamHI 

BauI 

5’...CA C G A G...3’ 

3’...G T G C T C...5’ 

#ER1841 200 u 




Concentration 

10 u/μl 

50 u/μl, HC 


1X Buffer BamHI at 37°C. 


BamHI is supplied in: 

 


 

BanI Thermo Scientific enzymes FastDigest BanI and BshNI 

BanII Thermo Scientific enzyme Eco24I 

20’ 

Concentration 

10 u/μl 




BauI is supplied in: 

 

1 mM DTT, 1 mM EDTA, 0.15% Triton X-100, 

 

BbeI Thermo Scientific enzymes FastDigest EheI and EheI 

Thermo Scientific enzyme SspDI 

BbrPI Thermo Scientific enzymes FastDigest PmlI and Eco72I 

BbsI Thermo Scientific enzymes FastDigest BbsI and BpiI 

BbuI Thermo Scientific enzymes FastDigest SphI and PaeI 

BbvI Thermo Scientific enzymes FastDigest BbvI and Lsp1109I ; 

FastDigest BseXI and BseXI 

BbvII Thermo Scientific enzymes FastDigest BbsI and BpiI 

BciVI Thermo Scientific enzyme BfuI 



20-50* 100 20-50 50-100* 100* 50-100 

* 









0-20 50-100 0-20 50-100 100 50-100 







DNA in 16 hours.

BclI 

5’...TG A T C A...3’ 

3’...A C T A G T...5’ 

#ER0721 


1000 u 



#ER0725 3000 u 

#ER0722 


5000 u 

10X Buffer G 2x1 ml 



FastDigest BclI 

BcnI 

5’...C CS G G...3’ 

3’...G G S C C...5’ 

#ER0061 1000 u 




FastDigest NciI 

BcuI 

5’...AC T A G T...3’ 

3’...T G A T C A...5’ 

#ER1251 400 u 

#ER1252 2000 u 




FastDigest SpeI 

Concentration 

10 u/μl 




BclI is supplied in: 

 


 

Concentration 

10 u/μl 



Concentration 

10 u/μl 

20’ 




BcuI is supplied in: 

 


 

BfaI Thermo Scientific enzymes FastDigest BfaI and FspBI 



20-50 100 20-50 20-50 100* 100 

* 





Note 

 

 


dam methylation. To avoid 

dam methylation, use a dam – , dcm – strain 




20-50 50-100 50-100 50-100 100 50-100 


BcnI is supplied in: 

 

200 mM NaCl, 1 mM EDTA, 1 mM DTT, 

 




 



50-100 50-100 0-20 20-50 100 0-20 







Adenovirus-2 DNA in 16 hours. 


90 

BfmI 

5’...CT R Y A G...3’ 

3’...G A Y R T C...5’ 

#ER1161 200 u 

#ER1162 1000 u 




FastDigest SfcI 

BfuI 

5’...G T A T C C(N) 6...3’ 

3’...C A T A G G(N) 5 ...5’ 

#ER1501 100 u 


10X Buffer BfuI 1 ml 



Concentration 

10 u/μl 



Concentration 

5 u/μl 


1X Buffer BfuI at 37°C. 


BfuI is supplied in: 

 

300 mM NaCl, 1 mM DTT, 1 mM EDTA, 

 



NR NR 0-20 0-20 NR 50-100* 

* 


Dam, Dcm, EcoKI, EcoBI, CpG – no effect. 

BfuAI Thermo Scientific enzymes FastDigest BspMI and BveI 

BfuCI Thermo Scientific enzymes FastDigest Sau3AI and Bsp143I ; 

FastDigest DpnI and DpnI 

FastDigest MboI and MboI 

20’ 



0-20 50-100 0-20 0-20 100 20-50 


BfmI is supplied in: 

 


 



BfoI Thermo Scientific enzyme FastDigest HaeII 

BfrI Thermo Scientific enzymes FastDigest AflII and BspTI 

BfrBI Thermo Scientific enzymes FastDigest NsiI and Mph1103I 

BglI 

5’...G C C N N N NN G G C...3’ 

3’...C G G N N N N N C C G...5’ 

#ER0071 


2000 u 



#ER0072 


5x2000 u 




FastDigest BglI 

20’ 

Concentration 

10 u/μl 




BglI is supplied in: 

 


 





Note 

 




0-20 50-100 100 100 0-20 100 




 




DNA in 16 hours.

BglII 

5’...AG A T C T...3’ 

3’...T C T A G A...5’ 

#ER0081 500 u 

#ER0082 2500 u 





FastDigest BglII 

5’...C CN G G...3’ 

3’...G G N C C...5’ 

#ER1421 500 u 

#ER1422 2500 u 





FastDigest ScrFI 

Concentration 

10 u/μl 




BglII is supplied in: 

 


 

BinI Thermo Scientific enzyme BspPI 

BlnI Thermo Scientific enzymes FastDigest AvrII and XmaJI 

BlpI Thermo Scientific enzymes FastDigest BlpI and Bpu1102I 

Bme1390I 

Concentration 

10 u/μl 




Bme1390I is supplied in: 

 


 

Bme1580I Thermo Scientific enzymes FastDigest Bme1580I and BseSI 

BmgBI Thermo Scientific enzyme AjiI 

BmsI Thermo Scientific enzyme FastDigest SfaNI 

BmtI Thermo Scientific enzymes FastDigest BmtI and BspOI ; 

FastDigest NheI and NheI 

BmyI Thermo Scientific enzymes FastDigest Bsp1286I and SduI 



0-20 20-50 100 50-100 0-20 100 



EcoBI: may overlap – cleavage impaired 

 







20-50 50-100 100 50-100 50-100 50-100 



Dcm, CpG may overlap – blocked 

 

Note 

dcm 

methylation. To avoid dcm methylation, use 



92 

BoxI 

5’...G A C N NN N G T C...3’ 

3’...C T G N N N N C A G...5’ 

#ER1431 500 u 




FastDigest PshAI 

BpiI 

5’...G A A G A C(N) 2...3’ 

3’...C T T C T G(N) 6 ...5’ 

#ER1011 200 u 

#ER1012 1000 u 





FastDigest BbsI 

BplI 

5’... 8(N)G A G(N) 5C T C(N) 13...3’ 

3’... 13(N)C T C(N) 5G A G(N) 8 ...5’ 

#ER1311 


100 u 



#ER1312 


500 u 


50X SAM 2x0.1 ml 


FastDigest BpII 


Concentration 

10 u/μl 




BoxI is supplied in: 

 


0.15% Triton X-100, 0.2 mg/ml BSA and 

 

Concentration 

10 u/μl 

20’ 




BpiI is supplied in: 

 


 

20’ 

Concentration 

5 u/μl 



SAM 0.05 mM. 


BplI is supplied in: 

 


 





0-20 0-20 0-20 20-50 100 20-50 











20-50 100 50-100 50-100 50-100 50-100 










0-20 20-50 0-20 0-20 100 20-50 





Note 

2+ for its activity, but 

is stimulated by S-adenosylmethionine. 

0.05 mM S-adenosylmethionine gives more 

than a 100-fold increase in BplI activity. Still, 

complete cleavage of some substrates with 

BplI is difficult to achieve. 

 



observed with further enzyme increase.

BpmI Thermo Scientific enzymes FastDigest BpmI and GsuI 

Bpu10I 

5’...C CT N A G C...3’ 

3’...G G A N T C G...5’ 

#ER1181 200 u 


10X Buffer Bpu10I 1 ml 



FastDigest Bpu10I 

Bpu1102I 

5’...G CT N A G C...3’ 

3’...C G A N T C G...5’ 

#ER0091 200 u 

#ER0092 1000 u 




FastDigest BlpI 

Concentration 

5 u/μl 


1X Buffer Bpu10I at 37°C. 


Bpu10I is supplied in: 

 


 

Concentration 

10 u/μl 




Bpu1102I is supplied in: 

 


 

BpuAI Thermo Scientific enzymes FastDigest BbsI and BpiI 

BsaI Thermo Scientific enzymes FastDigest Eco31I and Eco31I 

BsaAI Thermo Scientific enzymes FastDigest BsaAI and Ppu21I 

BsaBI Thermo Scientific enzymes FastDigest BsaBI and BseJI 

BsaHI Thermo Scientific enzymes FastDigest BsaHI and Hin1I 

BsaJI Thermo Scientific enzymes FastDigest BsaJI and BseDI 

BsaMI Thermo Scientific enzymes FastDigest Mva1269I and Mva1269I 

BseAI Thermo Scientific enzymes FastDigest Kpn2I and Kpn2I 



0-20 20-50* 50-100* 100* 50-100* 100* 

* 



Note 

 

of its recognition sequence are required. 

 

may result in incomplete DNA cleavage. 

Therefore, we recommend increasing the 

incubation time instead of using an excess 

of Bpu10I. 



50-100 50-100 20-50 20-50 100 20-50 









94 

BseDI 

5’...CC N N G G...3’ 

3’...G G N N C C...5’ 

#ER1081 300 u 

#ER1082 1500 u 




FastDigest BsaJI 

BseGI 

5’...G G A T G N N...3’ 

3’...C C T A C N N ...5’ 

#ER0871 500 u 

#ER0872 2500 u 




FastDigest BseGI 

BseJI 

5’...G A T N NN N A T C...3’ 

3’...C T A N N N N T A G...5’ 

#ER1711 2000 u 





FastDigest BsaBI 


Concentration 

10 u/μl 




BseDI is supplied in: 

 


 

Concentration 

10 u/μl 




BseGI is supplied in: 

 


 

Concentration 

10 u/μl 




 

 


 



50-100 20-50 0-20 0-20 100 50-100 



Note 

Incubation at 37°C results in 10% activity. 



20-50 50-100 20-50 20-50 100 20-50 




Note 




NR 100* 100 NR NR 100* 

* 





Note 

 

10% activity. 

 

 

dam 


a dam – , dcm – strain such as GM2163.

BseLI 

5’...C C N N N N NN N G G...3’ 

3’...G G N N N N N N N C C...5’ 

#ER1201 500 u 




FastDigest BslI 

BseMI 

5’...G C A A T G N N...3’ 

3’...C G T T A C N N ...5’ 

#ER1261 100 u 

#ER1262 500 u 





FastDigest BsrDI 

BseMII 

5’...C T C A G(N) 10...3’ 

3’...G A G T C(N) 8 ...5’ 

#ER1401 100 u 





FastDigest BspCNI 

Concentration 

10 u/μl 




BseLI is supplied in: 

 


 

Concentration 

5 u/μl 




BseMI is supplied in: 

 


 

Concentration 

1 u/μl 



SAM 0.01 mM. 


BseMII is supplied in: 

 


 



20-50 100 50-100 20-50 100 50-100 




 

Note 

 

dcm 

methylation. To avoid dcm methylation, use a 




0-20 20-50 0-20 100 50-100 50-100 




Note 




50-100 50-100 50-100 50-100 100 50-100 



Note 

 

 

Sinefungin can replace SAM in the restriction 

reaction. In this case DNA is not methylated 

and more than 95% of the ligated BseMII 

fragments can be recut by this enzyme. 


96 

BseNI 

5’...A C T G G N...3’ 

3’...T G A C C N ...5’ 

#ER0881 


1000 u 

10X Buffer B 1 ml 


#ER0882 


5000 u 




FastDigest BseNI 


Concentration 

10 u/μl 




BseNI is supplied in: 

 


 

BsePI Thermo Scientific enzymes FastDigest BssHII and PauI 

BseSI 

5’...G K G C MC...3’ 

3’...C M C G K G...5’ 

#ER1441 500 u 






BseXI 

5’...G C A G C(N) 8...3’ 

3’...C G T C G(N) 12 ...5’ 

#ER1451 100 u 

#ER1452 500 u 


10X Buffer BseXI 1 ml 



Concentration 

10 u/μl 




BseSI is supplied in: 

 


 

Concentration 

3 u/μl 


1X Buffer BseXI at 65°C. 


BseXI is supplied in: 

 


 



100 20-50 0-20 0-20 50-100 20-50 




Note 

 

activity. 



20-50 100 0-20 20-50 50-100 0-20 



EcoKI: may overlap – cleavage impaired 

 





Note 

 



NR NR NR NR NR NR 




Note 

 

 

BseXI may result in star activity. 

 

DNA. This may cause DNA band shifting during 

electrophoresis. To avoid atypical DNA band 

patterns, use the 6X DNA Loading Dye & SDS 

 

heat the digested DNA in the presence of SDS 

prior to electrophoresis.

BseYI Thermo Scientific enzyme FastDigest PspFI 

Bsh1236I 

5’...C GC G...3’ 

3’...G C G C...5’ 

#ER0921 500 u 

#ER0922 2500 u 





FastDigest Bsh1236I 

Bsh1285I 

5’...C G R YC G...3’ 

3’...G C Y R G C...5’ 

#ER0891 600 u 





FastDigest BsiEI 

BshNI 

5’...GG Y R C C...3’ 

3’...C C R Y G G...5’ 

#ER1001 2000 u 





FastDigest BanI 

Concentration 

10 u/μl 




Bsh1236I is supplied in: 

 


 

Concentration 

10 u/μl 

Concentration 

10 u/μl 

20’ 




Bsh1285I is supplied in: 

 


 

20’ 




BshNI is supplied in: 

 


 



0-20 0-20 50-100 100 20-50 50-100 






20-50 100 20-50 0-20 0-20 20-50 


Dam: may overlap – cleavage impaired 

 







Note 

 

overlapping dam methylation. To avoid dam 


as GM2163. 



0-20 20-50 100 50-100 0-20 100 


Dam, EcoBI – no effect. 


 






Note 

 




98 

BshTI 

5’...AC C G G T...3’ 

3’...T G G C C A...5’ 

#ER1461 200 u 

#ER1462 1000 u 





FastDigest AgeI 


Concentration 

10 u/μl 




BshTI is supplied in: 

 


 



0-20 20-50 100 50-100 20-50 20-50 









BsiEI Thermo Scientific enzymes FastDigest BsiEI and Bsh1285I 

BsiHKAI Thermo Scientific enzymes FastDigest Alw21I and Alw21I 

BsiWI Thermo Scientific enzymes FastDigest BsiWI and Pfl23II 

BsiYI Thermo Scientific enzymes FastDigest BslI and BseLI 

BslI Thermo Scientific enzymes FastDigest BslI and BseLI 

BsmI Thermo Scientific enzymes FastDigest Mva1269I and Mva1269I 

BsmAI Thermo Scientific enzymes FastDigest Alw26I and Alw26I 

BsmBI Thermo Scientific enzymes FastDigest BsmBI and Esp3I 

BsmFI Thermo Scientific enzymes FastDigest BsmFI and FaqI 

BsoBI Thermo Scientific enzymes FastDigest AvaI and Eco88I 

Bsp68I 

5’...T C GC G A...3’ 

3’...A G C G C T...5’ 

#ER0111 800 u 





FastDigest NruI 

Concentration 

10 u/μl 

20’ 




Bsp68I is supplied in: 

 


 



0-20 20-50 100 50-100 20-50 50-100 








Note 

 

Bsp68I may result in star activity.

Bsp119I 

5’...T TC G A A...3’ 

3’...A A G C T T...5’ 

#ER0121 2500 u 





Bsp120I 

5’...GG G C C C...3’ 

3’...C C C G G G...5’ 

#ER0131 1500 u 






Bsp143I 

5’...G A T C ...3’ 

3’... C T A G ...5’ 

#ER0781 300 u 

#ER0782 1500 u 


10X Buffer Bsp143I 1 ml 



FastDigest Sau3Al 

Concentration 

10 u/μl 





 


 

Concentration 

10 u/μl 





 


 

Concentration 

10 u/μl 

Bsp1286I Thermo Scientific enzymes FastDigest Bsp1286I and SduI 

20’ 


1X Buffer Bsp143I at 37°C. 



 


 



20-50 0-20 0-20 0-20 100 100 











100 20-50 0-20 20-50 50-100 0-20 




 





Note 

dcm 





20-50 20-50 0-20 0-20 50-100 20-50 




Note 

 


 


100 

Bsp1407I 

5’...TG T A C A...3’ 

3’...A C A T G T...5’ 

#ER0931 300 u 

#ER0932 1500 u 






20’ 

Concentration 

10 u/μl 





 


 

BspCNI Thermo Scientific enzymes FastDigest BspCNI and BseMII 

BspDI Thermo Scientific enzymes FastDigest ClaI and Bsu15I 

BspEI Thermo Scientific enzymes FastDigest Kpn2I and Kpn2I 

BspHI Thermo Scientific enzymes FastDigest BspHI and PagI 

BspLI 

5’...G G NN C C...3’ 

3’...C C N N G G...5’ 

#ER1151 200 u 

#ER1152 1000 u 




FastDigest NlaIV 

Concentration 

10 u/μl 

20’ 




BspLI is supplied in: 

 


 

BspLU11I Thermo Scientific enzyme PscI 

BspMI Thermo Scientific enzymes FastDigest BspMI and BveI 

BspMII Thermo Scientific enzymes FastDigest Kpn2I and Kpn2I 

BspOI 

5’...G C T A GC...3’ 

3’...C G A T C G...5’ 

#ER2041 200 u 





FastDigest BmtI 

Concentration 

10 u/μl 




BspOI is supplied in: 

 

500 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 


 



0-20 20-50 0-20 20-50 100 50-100 









50-100 50-100 0-20 20-50 100 20-50 




 

Note 

 





0-20 0-20 100 100 0-20 20-50 






lambda DNA in 16 hours.

BspPI 

5’...G G A T C(N) 4...3’ 

3’...C C T A G(N) 5 ...5’ 

#ER1321 100 u 

#ER1322 500 u 



5’...CT T A A G...3’ 

3’...G A A T T C...5’ 

#ER0831 1000 u 





FastDigest AflII 

Concentration 

2 u/μl 




BspPI is supplied in: 

 


 

BspQI Thermo Scientific enzymes FastDigest SapI and LguI 

BspTI 

Concentration 

10 u/μl 

20’ 




BspTI is supplied in: 

 


 



20-50 20-50 0-20 0-20 100 0-20 




Note 

 

dam 

methylation. To avoid dam methylation, use a 




0-20 0-20 100 20-50 0-20 50-100 







BspT104I Thermo Scientific enzymes FastDigest Bsp119I and Bsp119I 

BspT107I Thermo Scientific enzymes FastDigest BanI and BshNI 

BsrI Thermo Scientific enzymes FastDigest BseNI and BseNI 

BsrBI Thermo Scientific enzymes FastDigest BsrBI and MbiI 

BsrDI Thermo Scientific enzymes FastDigest BsrDI and BseMI 

BsrFI Thermo Scientific enzymes FastDigest BsrFI and Cfr10I 

BsrGI Thermo Scientific enzymes FastDigest Bsp1407I and Bsp1407I 

BsrSI Thermo Scientific enzymes FastDigest BseNI and BseNI 

BssHII Thermo Scientific enzyme FastDigest BssHII and PauI 

BssKI Thermo Scientific enzymes FastDigest ScrFI and Bme1390I 

BssSI Thermo Scientific enzyme BauI 

Bst98I Thermo Scientific enzymes FastDigest AflII and BspTI (AflII) 


102 

Bst1107I 

5’...G T AT A C...3’ 

3’...C A T A T G...5’ 

#ER0701 500 u 





FastDigest BstZ17I 


Concentration 

10 u/μl 




Bst1107I is supplied in: 

 


 

BstBI Thermo Scientific enzymes FastDigest Bsp119I and Bsp119I 

BstEII Thermo Scientific enzymes FastDigest Eco91I and Eco91I 

BstF5I Thermo Scientific enzymes FastDigest BseGI and BseGI ; 

Thermo Scientific enzyme FastDigest FokI 

BstNI Thermo Scientific enzymes FastDigest MvaI and MvaI ; 

EcoRII 

BstOI Thermo Scientific enzymes FastDigest MvaI and MvaI ; 

EcoRII 

BstPI Thermo Scientific enzymes FastDigest Eco91I and Eco91I 

BstUI Thermo Scientific enzymes FastDigest Bsh1236I and Bsh1236I 

BstXI 

5’...C C A N N N N NN T G G...3’ 

3’...G G T N N N N N N A C C...5’ 

#ER1021 500 u 

#ER1022 2500 u 





FastDigest BstXI 

Concentration 

10 u/μl 




BstXI is supplied in: 

 


 

BstYI Thermo Scientific enzymes FastDigest PsuI and PsuI 

BstZI Thermo Scientific enzymes FastDigest EagI and Eco52I 

BstZ17I Thermo Scientific enzymes FastDigest BstZ17I and Bst1107I 



20-50 50-100 100 100 20-50 100 




 







20-50 100 100 50-100 50-100 100 



Dcm: may overlap – cleavage impaired 

 





Note 

 

 

BstXI may result in star activity. 

dcm 


dam – , dcm – strain such as GM2163.

Bsu15I 

5’...A TC G A T...3’ 

3’...T A G C T A...5’ 

#ER0141 600 u 

#ER0145 


1000 u 


#ER0142 


3000 u 



FastDigest ClaI 

Bsu36I Thermo Scientific enzymes FastDigest Bsu36I and Eco81I 

BsuRI 

5’...G GC C...3’ 

3’...C C G G...5’ 

#ER0151 3000 u 


10X Buffer R 2x1 ml 



FastDigest HaeIII 

Concentration 

10 u/μl 

Concentration 

10 u/μl 

20’ 




Bsu15I is supplied in: 

 


 



BtrI Thermo Scientific enzyme AjiI 

BtsCI Thermo Scientific enzymes FastDigest BseGI and BseGI ; 

Thermo Scientific enzyme FastDigest FokI 



20-50 20-50 20-50 20-50 100 20-50 



Dcm, EcoKI – no effect. 







Note 

dam 





20-50 20-50 50-100 100 50-100 100 


BsuRI is supplied in: 

 


50% glycerol. 




104 

BveI 

5’...A C C T G C (N) 4 ...3’ 

3’...T G G A C G (N) 8 ...5’ 

#ER1741 250 u 




50X oligonucleotide 2X25 μl 


FastDigest BspMI 

Note 

 

are required for efficient cleavage. 

 

BveI recognition sequence in the reaction 

mixture significantly improves cleavage of 

plasmid DNAs, especially of those with a single 

CaiI 

5’...C A G N N NC T G...3’ 

3’...G T C N N N G A C...5’ 

#ER1391 500 u 




FastDigest AlwNI 


Concentration 

5 u/μl 




BveI site. Still, complete cleavage of some 

substrates with BveI is difficult to achieve. 

 




 

Concentration 

10 u/μl 

20’ 




CaiI is supplied in: 

 


50% glycerol. 



0-20 20-50 100 20-50 50-100 100 


BveI is supplied in: 

 


 





cleaved DNA. This may cause DNA band 

shifting during electrophoresis. To avoid 

atypical DNA band patterns, use the 6X DNA 

 

sample preparation or heat the digested 

DNA in the presence of SDS prior to 




20-50 20-50 20-50 50-100 100 50-100 









Note 

CaiI is blocked by overlapping dcm methylation. 



CauII Thermo Scientific enzymes FastDigest NciI and BcnI 

CelII Thermo Scientific enzymes FastDigest BlpI and Bpu1102I 

CfoI Thermo Scientific enzymes FastDigest HinP1I and Hin6I 

FastDigest HhaI and HhaI 

20’

Cfr9I 

5’...CC C G G G...3’ 

3’...G G G C C C...5’ 

#ER0171 300 u 

#ER0172 1500 u 


10X Buffer Cfr9I 1 ml 


Cfr10I 

5’...RC C G G Y...3’ 

3’...Y G G C C R...5’ 

#ER0181 200 u 


10X Buffer Cfr10I 1 ml 



FastDigest BsrFI 

Cfr13I 

5’...GG N C C...3’ 

3’...C C N G G...5’ 

#ER0191 1000 u 





20’ 

Concentration 

10 u/μl 


1X Buffer Cfr9I at 37°C. 


Cfr9I is supplied in: 

 


 

Concentration 

10 u/μl 


1X Buffer Cfr10I at 37°C. 



 

100 mM KCl, 1 mM DTT, 1 mM EDTA, 

0.2 mg/ml BSA and 50% glycerol. 

Concentration 

10 u/μl 





 


 

20’ 



0-20 0-20 0-20 0-20 20-50 0-20 




 





Note 

 

with Cfr9I, the concentration of DNA should 

be no less than 50 μg/ml in the reaction 

buffer. 



0-20 20-50 20-50 50-100* 20-50 50-100 

* 








Note 

 

Cfr10I may result in star activity. 

 




50-100 50-100 20-50 20-50 100 20-50 




 

Note 

Cfr13I is blocked by overlapping dcm 




106 

Cfr42I 

5’...C C G CG G...3’ 

3’...G G C G C C...5’ 

#ER0201 1200 u 

#ER0205 


2000 u 



#ER0202 


5x1200 u 



ClaI Thermo Scientific enzymes FastDigest ClaI and Bsu15I 

CpoI 

5’...C GG W C C G...3’ 

3’...G C C W G G C...5’ 

#ER0741 200 u 

#ER0742 1000 u 




FastDigest RsrII 

CseI 

5’...G A C G C (N) 5...3’ 

3’...C T G C G (N) 10 ...5’ 

#ER1901 100 u 





FastDigest HgaI 


20’ 

Concentration 

10 u/μl 





 


 

Concentration 

10 u/μl 

20’ 



Concentration 

5 u/μl 




CseI is supplied in: 

 

1 mM DTT, 0.1 mM EDTA and 

50% glycerol. 



100 50-100 0-20 0-20 50-100 0-20 








Note 

 

to cleave with Cfr42I, the same as with its 

prototype SacII. At least two copies of Cfr42I 

recognition site are required for efficient 

cleavage. 



20-50 50-100 50-100 20-50 100 50-100 


CpoI is supplied in: 

 


 






NR 50-100* 50-100 100 100* 50-100 

* 





Note 

 

recognition sequence are required. 

 


 




 


presence of SDS prior to electrophoresis.

CsiI Thermo Scientific enzyme FastDigest SexAI 

CspI Thermo Scientific enzymes FastDigest RsrII and CpoI 

Csp6I 

5’...GT A C...3’ 

3’...C A T G...5’ 

#ER0211 1500 u 





FastDigest Csp6I 

20’ 

Concentration 

10 u/μl 





100 50-100 0-20 0-20 50-100 0-20 


Csp6I is supplied in: 

 


 



Csp45I Thermo Scientific enzymes FastDigest Bsp119I and Bsp119I 

CviAII Thermo Scientific enzymes FastDigest NlaIII and Hin1II 

CviQI Thermo Scientific enzymes FastDigest Csp6I and Csp6I 

DdeI Thermo Scientific enzymes FastDigest DdeI and HpyF3I 

DpnI 

5’...G m6A T C...3’ 

3’...C T m6A G...5’ 

#ER1701 500 u 

#ER1705 1000 u 

#ER1702 2500 u 




FastDigest DpnI 

Concentration 

10 u/μl 




DpnI is supplied in: 

 


 


Dam: does not cut dam – Activity in Five Buffer System, % 


100 100 50-100 50-100 100 50-100 

DNA 



Note 

 

within the recognition sequence to cleave DNA. 

dam + strain will be a 

substrate for DpnI. 

 

dam sites. Hemi-adenomethylated dam sites 

DpnI cleaves 60X more slowly. 

 


 

DpnII Thermo Scientific enzymes FastDigest DpnI and DpnI 

FastDigest Sau3AI and Bsp143 

FastDigest MboI and MboI 


108 

DraI 

5’...T T TA A A...3’ 

3’...A A A T T T...5’ 

#ER0221 1500 u 



#ER0223 HC, 7500 u 




FastDigest DraI, 


20’ 

Concentration 

10 u/μl, 

50 u/μl, HC 




DraI is supplied in: 

 


 

DraII Thermo Scientific enzymes FastDigest Eco0109I and EcoO109I 

DraIII Thermo Scientific enzymes FastDigest DraIII and AdeI 

DrdI Thermo Scientific enzymes FastDigest DrdI 

EagI Thermo Scientific enzymes FastDigest EagI and Eco52I 

Eam1104I 

5’...C T C T T C(N) 1...3’ 

3’...G A G A A G(N) 4 ...5’ 

#ER0231 300 u 

#ER0232 1500 u 




FastDigest EarI 

Eam1105I 

5’...G A C N N NN N G T C...3’ 

3’...C T G N N N N N C A G...5’ 

#ER0241 1000 u 


10X Buffer Eam1105I 1 ml 



FastDigest Eam1105I 

Concentration 

10 u/μl 

20’ 




Eam1104I is supplied in: 

 


 

20’ 

Concentration 

10 u/μl 


1X Buffer Eam1105I at 37°C. 


Eam1105I is supplied in: 

 


 



50-100 50-100 20-50 20-50 100 50-100 










50-100 50-100 0-20 0-20 100 0-20 








Note 

DNA are difficult to 

cleave with Eam1104I, the same as with its 

prototype Ksp632I. 



20-50 50-100 0-20 0-20 50-100 20-50 






DNA in 16 hours.

EarI Thermo Scientific enzymes FastDigest EarI and Eam1104I 

Ecl136II 

5’...G A GC T C...3’ 

3’...C T C G A G...5’ 

#ER0251 1500 u 


10X Buffer Ecl136II 1 ml 




5’...G R G C YC...3’ 

3’...C Y C G R G...5’ 

#ER0281 1500 u 



20’ 

Concentration 

10 u/μl 


1X Buffer Ecl136II at 37°C. 


Ecl136II is supplied in: 

 


 

EclHKI Thermo Scientific enzymes FastDigest Eam1105I and Eam1105I 

EclXI Thermo Scientific enzymes FastDigest EagI and Eco52I 

Eco24I 

20’ 

Concentration 

10 u/μl 




Eco24I is supplied in: 

 


 



50-100 20-50 0-20 0-20 50-100 0-20 




 








50-100 50-100 0-20 20-50 100 0-20 









110 

Eco31I 

5’...G G T C T C(N) 1...3’ 

3’...C C A G A G(N) 5 ...5’ 

#ER0291 


1000 u 



#ER0292 


5000 u 





Eco32I 

5’...G A TA T C...3’ 

3’...C T A T A G...5’ 

#ER0301 


2000 u 

10X Buffer R 1ml 

10X Buffer Tango 1ml 

#ER0305 4000 u 

#ER0302 5x2000 u 

#ER0303 HC, 10,000 u 





FastDigest EcoRV 

Eco47I 

5’...GG W C C...3’ 

3’...C C W G G...5’ 

#ER0311 


800 u 



#ER0312 


4000 u 






Concentration 

10 u/μl 





 


 

Concentration 

10 u/μl 

50 u/μl, HC 





 


 

Concentration 

10 u/μl 

20’ 

20’ 





50-100 100 0-20 0-20 50-100 20-50 




 






Note 

 





0-20 50-100 50-100 100 20-50 100 










0-20 50-100 50-100 100 50-100 50-100 



 


 




Note 

dcm 



Eco47III 

5’...A G CG C T...3’ 

3’...T C G C G A...5’ 

#ER0321 200 u 

#ER0322 1000 u 





FastDigest AfeI 

Eco52I 

5’...CG G C C G...3’ 

3’...G C C G G C...5’ 

#ER0331 500 u 

#ER0332 2500 u 


10X Buffer Eco52I 1 ml 



FastDigest EagI 

Concentration 

10 u/μl 

20’ 




Eco47III is supplied in: 

 

1 mM DTT, 1mM EDTA, 0.2 mg/ml BSA and 

 

Concentration 

10 u/μl 


1X Buffer Eco52I at 37°C. 



 


 

Eco53kI Thermo Scientific enzymes FastDigest Ecl136II and Ecl136II ; 

Thermo Scientific enzymes FastDigest SacI and SacI 

20’ 



0-20 20-50 100 100 50-100 100 











0-20 0-20 0-20 20-50 0-20 20-50 









112 

Eco57I 

5’...C T G A A G(N) 16...3’ 

3’...G A C T T C(N) 14 ...5’ 

#ER0341 


200 u 




#ER0342 


1000 u 


50X SAM 2x0.1 ml 



FastDigest AcuI 

Eco72I 

5’...C A CG T G...3’ 

3’...G T G C A C...5’ 

#ER0361 2000 u 




FastDigest PmlI 


Concentration 

5 u/μl 

20’ 



SAM 0.01 mM. 

Note 

2+ for its activity, but is 

stimulated by S-adenosylmethionine. 0.01 mM 

S-adenosylmethionine gives a 100-fold 

increase in Eco57I activity. Still, complete 

cleavage of some substrates with Eco57I is 

difficult to achieve. 

 

its recognition sequence are required. 

 




Concentration 

10 u/μl 





 


 

20’ 



100 100 20-50 20-50 50-100 50-100 



 

100 mM NaCl, 1 mM EDTA, 1 mM DTT and 

 




 

the cleaved DNA. This may cause DNA 


avoid atypical DNA band patterns, use 

the 6X DNA Loading Dye & SDS Solution 

 

the digested DNA in the presence of SDS 

prior to electrophoresis. 



NR NR 0-20 0-20 100 20-50 









Note 

 

Eco72I may result in star activity.

Eco81I 

5’...C CT N A G G...3’ 

3’...G G A N T C C...5’ 

#ER0371 500 u 

#ER0372 2500 u 




FastDigest Bsu36l 

Eco88I 

5’...CY C G R G...3’ 

3’...G R G C Y C...5’ 

#ER0381 1000 u 




FastDigest AvaI 

Eco91I 

5’...GG T N A C C...3’ 

3’...C C A N T G G...5’ 

#ER0391 


1000 u 



#ER0392 


5000 u 





Concentration 

10 u/μl 





 


 

Concentration 

10 u/μl 

20’ 





 


 

20’ 

Concentration 

10 u/μl 





 


 



50-100 100 0-20 0-20 100 0-20 










100 50-100 0-20 0-20 100 20-50 




 







20-50 20-50 100 50-100 NR 100 









114 

Eco105I 

5’...T A CG T A...3’ 

3’...A T G C A T...5’ 

#ER0401 


600 u 


#ER0402 


3000 u 



FastDigest SnaBI 

Eco130I 

5’...CC W W G G...3’ 

3’...G G W W C C...5’ 

#ER0411 2500 u 





FastDigest StyI 

Eco147I 

5’...A G GC C T...3’ 

3’...T C C G G A...5’ 

#ER0421 


1000 u 



#ER0422 


5000 u 




FastDigest StuI 


Concentration 

10 u/μl 





 

1 mM DTT, 1 mM EDTA, 

1 mM phenylmethylsulfonylfluoride, 

 

Concentration 

10 u/μl 

20’ 





 


 

Concentration 

10 u/μl 





 


 

EcoICRI Thermo Scientific enzymes FastDigest Ecl136II and Ecl136II ; 

FastDigest SacI and SacI 

EcoNI Thermo Scientific enzymes FastDigest EcoNI and XagI 

EcoO65I Thermo Scientific enzymes FastDigest Eco91I and Eco91I 



100* 50-100 0-20 0-20 100 0-20 

* 








Note 

 

Eco105I may result in star activity. 



0-20 20-50 100 50-100 50-100 100 









100 50-100 20-50 20-50 50-100 0-20 









Note 

dcm 



EcoO109I 

5’...R GG N C C Y...3’ 

3’...Y C C N G G R...5’ 

#ER0261 2000 u 




FastDigest EcoO109I 

EcoRI 

5’...GA A T T C...3’ 

3’...C T T A A G...5’ 

#ER0271 


5000 u 

10X Buffer EcoRI 2x1 ml 


#ER0275 


10,000 u 



#ER0272 5x5000 u 

#ER0273 HC, 25,000 u 





FastDigest EcoRI 

20’ 

Concentration 

10 u/μl 




EcoO109I is supplied in: 

 


 

20’ 

Concentration 

10 u/μl 

50 u/μl, HC 


1X Buffer EcoRI at 37°C. 


EcoRI is supplied in: 

 


0.2 mg/ml BSA, 0.15% Triton X-100 and 

 



50-100 20-50 20-50 20-50 100 100 






Minimum 5 units of the enzyme is required 



Note 

dcm 





0-20 NR 100 100* NR 100 

* 




 






116 

EcoRII 

5’...C C W G G ...3’ 

3’... G G W C C ...5’ 

#ER1921 200 u 




EcoRV Thermo Scientific enzymes FastDigest EcoRV and Eco32I 

EcoT14I Thermo Scientific enzymes FastDigest StyI and Eco130I 

EcoT22I Thermo Scientific enzymes FastDigest NsiI and Mph1103I 

EheI 

5’...G G CG C C...3’ 

3’...C C G C G G...5’ 

#ER0441 500 u 





EspI Thermo Scientific enzymes FastDigest BlpI and Bpu1102I 


Concentration 

10 u/μl 




EcoRII is supplied in: 

 

1 mM DTT, 1mM EDTA, 0.2 mg/ml BSA and 

 



Dcm: completely overlaps – blocked 

20’ 

Concentration 

10 u/μl 




EheI is supplied in: 

 


 



20-50 50-100 100 50-100 20-50 50-100 

Note 

 

site are required for efficient cleavage. For 

cleavage of DNA substrates with only one 

 

neoschizomer of EcoRII, is recommended. 

 

DNA. This may cause DNA band shifting during 

electrophoresis. To avoid atypical DNA band 

patterns, use the 6X DNA Loading Dye & SDS 

 

heat the digested DNA in the presence of SDS 

prior to electrophoresis. 

dcm 





20-50 50-100 0-20 0-20 100 20-50 








Note 

and 

pBR322 DNAs.

Esp3I 

5’...C G T C T C(N) 1...3’ 

3’...G C A G A G(N) 5 ...5’ 

#ER0451 200 u 

#ER0452 1000 u 




FastDigest BsmBI 

FaqI 

5’...G G G A C(N) 10...3’ 

3’...C C C T G(N) 14 ...5’ 

#ER1811 100 u 





FastDigest BsmFI 

Concentration 

10 u/μl 



 


Esp3I is supplied in: 

 


 

Concentration 

2 u/μl 



SAM 0.05 mM. 


FaqI is supplied in: 

 


 




FatI Thermo Scientific enzymes FastDigest NlaIII and Hin1II 

FbaI Thermo Scientific enzymes FastDigest BclI and BclI 

FinI Thermo Scientific enzymes FastDigest BsmFI and Faq 

FnuDII Thermo Scientific enzymes FastDigest Bsh1236I and Bsh1236I 

Fnu4HI Thermo Scientific enzymes FastDigest Fnu4HI and SatI 

FokI Thermo Scientific enzymes FastDigest FokI; 

FastDigest BseGI and BseGI 

FspI Thermo Scientific enzymes FastDigest FspI and NsbI 

20’ 


B +DTT G +DTT O +DTT R +DTT Tango +DTT 2X Tango +DTT 

100 20-50 0-20 0-20 100 0-20 









Note 

 

Freshly made DTT solution should be added 

to the reaction buffer. 



20-50 20-50 0-20 0-20 100 20-50 

Note 



0.05 mM S-adenosylmethionine gives more 

than a 2-fold increase in FaqI activity. Still, 

complete cleavage of some substrates is 

difficult to achieve. 

 


 




 




 




118 

FspAI 

5’...R T G CG C A Y...3’ 

3’...Y A C G C G T R...5’ 

#ER1661 100 u 

#ER1662 500 u 





FastDigest FspAI 

FspBI 

5’...CT A G...3’ 

3’...G A T C...5’ 

#ER1761 500 u 

#ER1762 2500 u 




FastDigest BfaI 


20’ 

Concentration 

5 u/μl 



Concentration 

10 u/μl 



GsaI Thermo Scientific enzyme FastDigest PspFI 

GsuI 

5’...C T G G A G(N) 16...3’ 

3’...G A C C T C(N) 14 ...5’ 

#ER0461 100 u 

#ER0462 500 u 





FastDigest BpmI 


FspBI is supplied in: 

 


 

Concentration 

5 u/μl 





0-20 0-20 100 50-100 0-20 50-100 


FspAI is supplied in: 

 


50% glycerol. 







50-100 20-50 0-20 0-20 100 0-20 

20’ 


GsuI is supplied in: 

 

1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 

 












100 50-100 20-50 20-50 100 50-100 

Note 

 



0.01 mM S-adenosylmethionine gives a 

2-fold increase in activity. 

 




 



dcm 


dam – , dcm – strain such as GM2163.

HaeII Thermo Scientific enzyme FastDigest HaeII 

HaeIII Thermo Scientific enzymes FastDigest HaeIII and BsuRI 

HapII Thermo Scientific enzymes FastDigest HpaII and HpaII; 

FastDigest MspI and MspI 

HgaI Thermo Scientific enzymes FastDigest HgaI and CseI 

HgiAI Thermo Scientific enzymes FastDigest Alw21I and Alw21I 

HgiCI Thermo Scientific enzymes FastDigest BanI and BshNI 

HgiJII Thermo Scientific enzyme Eco24I 

HhaI 

5’...G C GC...3’ 

3’...C G C G...5’ 

#ER1851 2000 u 




FastDigest HhaI 

Hin1I 

5’...G RC G Y C...3’ 

3’...C Y G C R G...5’ 

#ER0471 300 u 






Concentration 

10 u/μl 



Concentration 

10 u/μl 




Hin1I is supplied in: 

 


 

20’ 



50-100 50-100 20-50 20-50 100 20-50 


HhaI is supplied in: 

 


50% glycerol. 






20-50 100 20-50 20-50 20-50 20-50 



Dcm: may overlap – cleavage impaired 

 







Note 

 




120 

Hin1II 

5’... C A T G...3’ 

3’... G T A C ...5’ 

#ER1831 300 u 





FastDigest NlaIII 

Hin6I 

5’...GC G C...3’ 

3’...C G C G...5’ 

#ER0481 2000 u 





HinP1I Thermo Scientific enzymes FastDigest HhaI and HhaI 

FastDigest HinP1I and Hin6I 

HincII 

5’...G T YR A C...3’ 

3’...C A R Y T G...5’ 

#ER0491 500 u 

#ER0492 2500 u 




FastDigest HincII 


Concentration 

5 u/μl 

Concentration 

10 u/μl 

20’ 



Concentration 

10 u/μl 



HindII Thermo Scientific enzymes FastDigest HincII and HincII 

20’ 




Hin1II is supplied in: 

 



 

20’ 



50-100 100 20-50 50-100 50-100 50-100 




Note 

 

At -20°C the half-life of Hin1II is 6 months. 



50-100 50-100 20-50 20-50 100 50-100 


Hin6I is supplied in: 

 


 






50-100 50-100 20-50 50-100 100 50-100 


HincII is supplied in: 

 


 




 

EcoKI, EcoBI: may overlap – blocked

HindIII 

5’...AA G C T T...3’ 

3’...T T C G A A...5’ 

#ER0501 


5000 u 



#ER0505 


10,000 u 



#ER0502 5x5000 u 

#ER0503 HC, 25,000 u 





FastDigest HindIII 

HinfI 

5’...GA N T C...3’ 

3’...C T N A G...5’ 

#ER0801 


2000 u 



#ER0802 5x2000 u 

#ER0803 HC, 10,000 u 



10X Buffer Tango 

FastDigest HinfI 

1 ml 

Concentration 

10 u/μl 

50 u/μl, HC 




HindIII is supplied in: 

 


 

20’ 

Concentration 

10 u/μl 

50 u/μl, HC 



HpaI Thermo Scientific enzymes FastDigest HpaI and KspAI 

HpaII 

5’...CC G G...3’ 

3’...G G C C...5’ 

#ER0511 


1000 u 


#ER0512 


5000 u 




20’ 

Concentration 

10 u/μl 





0-20 20-50 0-20 100 50-100 50-100 



EcoBI: may overlap – cleavage impaired 

 







0-20 20-50 50-100 100 50-100 50-100 


HinfI is supplied in: 

 


 




 




50-100 50-100 0-20 20-50 100 20-50 


HpaII is supplied in: 

 


 




Note 

 

 

 


122 

HphI 

5’...G G T G A(N) 8...3’ 

3’...C C A C T(N) 7 ...5’ 

#ER1101 300 u 

#ER1102 1500 u 




Hpy8I 

5’...G T NN A C...3’ 

3’...C A N N T G...5’ 

#ER1571 200 u 

#ER1572 1000 u 




FastDigest Hpy8I 


20’ 

Concentration 

10 u/μl 




HphI is supplied in: 

 


 

Concentration 

10 u/μl 



HpyCH4III Thermo Scientific enzymes FastDigest TaaI and TaaI 

HpyCH4IV Thermo Scientific enzymes FastDigest TaiI and TaiI 

HpyF3I 

5’...CT N A G...3’ 

3’...G A N T C...5’ 

#ER1881 500 u 

#ER1882 2500 u 




FastDigest DdeI 

Concentration 

10 u/μl 

20’ 





100 0-20 0-20 0-20 20-50 0-20 


Dam, EcoBI: may overlap – blocked 


Note 

dam 





50-100 50-100 0-20 20-50 100 50-100 


Hpy8I is supplied in: 

 


 




 




20-50 20-50 20-50 20-50 100 50-100 


HpyF3I is supplied in: 

 


 



EcoBI: may overlap – effect not determined.

HpyF10VI 

5’...G C N N N N NN N G C...3’ 

3’...C G N N N N N N N C G...5’ 

#ER1731 300 u 

#ER1732 1500 u 




FastDigest HpyF10VI 

KpnI 

5’...G G T A CC...3’ 

3’...C C A T G G...5’ 

#ER0521 


4000 u 

10X Buffer KpnI 2x1 ml 


#ER0522 5x4000 u 

#ER0523 HC, 20,000 u 


10X Buffer KpnI 4x1 ml 



FastDigest KpnI 

Concentration 

10 u/μl 



Hsp92I Thermo Scientific enzymes FastDigest BsaHI and Hin1I 

Hsp92II Thermo Scientific enzymes FastDigest NlaIII and Hin1II 

ItaI Thermo Scientific enzymes FastDigest Fnu4HI and SatI 

KasI Thermo Scientific enzymes FastDigest EheI and EheI 


KflI Thermo Scientific enzyme FastDigest SanDI 

Kpn2I 

5’...TC C G G A...3’ 

3’...A G G C C T...5’ 

#ER0531 500 u 

#ER0532 2500 u 




FastDigest Kpn2I 

Concentration 

10 u/μl 

50 u/μl, HC 


1X Buffer KpnI at 37°C. 


KpnI is supplied in: 

 


 

Concentration 

10 u/μl 




Kpn2I is supplied in: 

 


 



0-20 0-20 0-20 0-20 100 50-100 


HpyF10VI is supplied in: 

 


 






20-50 0-20 0-20 0-20 20-50 0-20 









50-100 50-100 0-20 20-50 100 50-100 








Note 



124 

KspI Thermo Scientific enzyme Cfr42I 

Ksp632I Thermo Scientific enzymes FastDigest EarI and Eam1104I 

KspAI 

5’...G T TA A C...3’ 

3’...C A A T T G...5’ 

#ER1031 500 u 

#ER1032 2500 u 






LguI 

5’...G C T C T T C(N) 1...3’ 

3’...C G A G A A G(N) 4 ...5’ 

#ER1931 100 u 

#ER1932 500 u 




FastDigest SapI 

Lsp1109I 

5’...G C A G C(N) 8...3’ 

3’...C G T C G(N) 12 ...5’ 

#ER2071 200 u 


10X Buffer Lsp1109I 1 ml 





Concentration 

10 u/μl 

20’ 




KspAI is supplied in: 

 


 

Concentration 

5 u/μl 

20’ 




LguI is supplied in: 

 


 

Concentration 

5 u/μl 

20’ 


1X Buffer Lsp1109I at 37°C. 


Lsp1109I is supplied in: 

 


 



100 50-100* 20-50 20-50 100* 50-100 

* 




 








20-50 50-100 20-50 20-50 100 20-50 










0-20 20-50* 50-100* 100* 20-50* 20-50* 

* 




Note 

 

Lsp1109I may result in star activity. 

 




 



electrophoresis.

LweI 

5’...G C A T C(N) 5...3’ 

3’...C G T A G(N) 9 ...5’ 

#ER1621 100 u 

#ER1622 500 u 




FastDigest SfaNI 

Concentration 

10 u/μl 




LweI is supplied in: 

 


 

MaeI Thermo Scientific enzymes FastDigest BfaI and FspBI 

MaeII Thermo Scientific enzymes FastDigest TaiI and TaiI 

MamI Thermo Scientific enzymes FastDigest BsaBI and BseJI 

MauBI 

5’...C GC G C G C G...3’ 

3’...G C G C G C G C...5’ 

#ER2081 100 u 





FastDigest MauBI 

MbiI 

5’...C C GC T C...3’ 

3’...G G C G A G...5’ 

#ER1271 1000 u 





20’ 

20’ 

Concentration 

5 u/μl 




 

 

 

Concentration 

10 u/μl 




 

 

 

20’ 



0-20 0-20 0-20 20-50 100 20-50 


Dam, Dcm, EcoKI– no effect. 



Note 

 


 




 





0-20 0-20 0-20 0-20 100 0-20 










20-50 100 20-50 20-50 100 20-50 




 







126 

MboI 

5’...G A T C ...3’ 

3’... C T A G ...5’ 

#ER0811 300 u 

#ER0812 1500 u 






MboII 

5’...G A A G A(N) 8...3’ 

3’...C T T C T(N) 7 ...5’ 

#ER0821 300 u 

#ER0822 1500 u 





FastDigest MboII 


20’ 

Concentration 

10 u/μl 




 

 

 

Concentration 

5 u/μl 




MboII is supplied in: 

 


 




McrI Thermo Scientific enzymes FastDigest BsiEI and Bsh1285I 

MfeI Thermo Scientific enzymes FastDigest MfeI and MunI 

MflI Thermo Scientific enzymes FastDigest PsuI and PsuI 

MjaIV Thermo Scientific enzymes FastDigest Hpy8I and Hpy8I 

20’ 



50-100 50-100 50-100 100 50-100 100 





Note 

dam 

methylation. To avoid dam methylation, 


 


 



100 50-100 20-50 0-20 50-100 20-50 

Note 

dam 

methylation. To avoid dam methylation, 


 

MboII may result in star activity. 

 

a single-base 3’-extension which are 

more difficult to ligate than blunt-ended 

fragments. 

 




 



electrophoresis.

MlsI 

5’...T G GC C A...3’ 

3’...A C C G G T...5’ 

#ER1211 200 u 

#ER1212 1000 u 






MluI 

5’...AC G C G T...3’ 

3’...T G C G C A...5’ 

#ER0561 


1000 u 



#ER0562 


5000 u 




FastDigest MluI 

Concentration 

5 u/μ 

20’ 




MlsI is supplied in: 

 


 

Concentration 

10 u/μl 




MluI is supplied in: 

 


 

MluNI Thermo Scientific enzymes FastDigest MscI and MlsI 

MlyI Thermo Scientific enzymes FastDigest MlyI and SchI 

MnlI 

5’...C C T C(N) 7...3’ 

3’...G G A G(N) 6 ...5’ 

#ER1071 300 u 

#ER1072 1500 u 






20’ 

Concentration 

10 u/μl 




MnlI is supplied in: 

 


 



0-20 20-50 0-20 100 20-50 50-100 








Note 

 





0-20 20-50 50-100 100 20-50 50-100 










50-100 100 20-50 20-50 20-50 20-50 




Note 

 

a single-base 3’-extension which are 

more difficult to ligate than blunt-ended 

fragments. 

 




 





128 

Mph1103I 

5’...A T G C AT...3’ 

3’...T A C G T A...5’ 

#ER0731 


1000 u 



#ER0732 


5000 u 





MreI 

5’...C GC C G G C G...3’ 

3’...G C G G C C G C...5’ 

#ER2021 300 u 





FastDigest MreI 


Concentration 

10 u/μl 

20’ 



Concentration 

10 u/μl 



MroI Thermo Scientific enzymes FastDigest Kpn2I and Kpn2I 

MscI Thermo Scientific enzymes FastDigest MscI and MlsI 

MseI Thermo Scientific enzymes FastDigest Tru1I and Tru1I ; 

Thermo Scientific enzyme FastDigest MseI 

MslI Thermo Scientific enzymes FastDigest MslI and RseI 

MspI 

5’...CC G G...3’ 

3’...G G C C...5’ 

#ER0541 


3000 u 

10X Buffer Tango 2X1 ml 

#ER0542 


5x3000 u 




Concentration 

10 u/μl 




MspI is supplied in: 

 


 



0-20 50-100 20-50 100 50-100 50-100 


Mph1103I is supplied in: 

 


 






20-50 100 0-20 0-20 50-100 0-20 


MreI is supplied in: 

 

200 mM KCl, 0.1 mM EDTA, 


 






50-100 50-100 0-20 0-20 100 50-100 



Note 

 

the external C in the sequence CCGG is 

methylated, MspI and HpaII cannot cleave. 

However, unlike HpaII, MspI can cleave the 

sequence when the internal C residue is 

methylated.

MssI 

5’...G T T TA A A C...3’ 

3’...C A A A T T T G...5’ 

#ER1341 250 u 

#ER1342 1250 u 






MunI 

5’...CA A T T G...3’ 

3’...G T T A A C...5’ 

#ER0751 300 u 

#ER0752 1500 u 





FastDigest MfeI 

Concentration 

5 u/μl 




MssI is supplied in: 

 


 

MstI Thermo Scientific enzymes FastDigest FspI and NsbI 

MvaI 

5’...C CW G G...3’ 

3’...G G W C C...5’ 

#ER0551 2000 u 






Concentration 

10 u/μl 

20’ 

20’ 




MunI is supplied in: 

 


 

Concentration 

10 u/μl 




MvaI is supplied in: 

 


 



100 0-20 0-20 0-20 20-50 0-20 










100 100 0-20 0-20 100 0-20 










20-50 20-50 50-100 100 20-50* 100 

* 



Note 

 

not require multiple copies of recognition 

site for efficient cleavage. 


130 

Mva1269I 

5’...G A A T G C N...3’ 

3’...C T T A C G N ...5’ 

#ER0961 200 u 

#ER0962 1000 u 





FastDigest Mva1269I 


20’ 

Concentration 

10 u/μl 




Mva1269I is supplied in: 

 


 

MvnI Thermo Scientific enzymes FastDigest Bsh1236I and Bsh1236I 

MwoI Thermo Scientific enzymes FastDigest HpyF10VI and HpyF10VI 

NaeI Thermo Scientific enzymes FastDigest NaeI and PdiI 

NarI Thermo Scientific enzymes FastDigest EheI and EheI 


NciI Thermo Scientific enzymes FastDigest NciI and BcnI 

NcoI 

5’...CC A T G G...3’ 

3’...G G T A C C...5’ 

#ER0571 500 u 

#ER0575 1000 u 

#ER0572 2500 u 





20’ 

Concentration 

10 u/μl 




NcoI is supplied in: 

 


50% glycerol. 



0-20 20-50 50-100 100 0-20 50-100 










20-50 20-50 20-50 50-100 100 100 






DNA in 16 hours.

NdeI 

5’...C AT A T G...3’ 

3’...G T A T A C...5’ 

#ER0581 500 u 

#ER0582 


2500 u 



#ER0585 


4000 u 




FastDigest NdeI 

20’ 

Concentration 

10 u/μl 




NdeI is supplied in: 

 


 



0-20 0-20 100 50-100 0-20 50-100 







NdeII Thermo Scientific enzymes FastDigest Sau3AI and Bsp143I 


FastDigest MboI and MboI 

NgoMIV Thermo Scientific enzymes FastDigest NaeI and PdiI 

NheI 

5’...GC T A G C...3’ 

3’...C G A T C G...5’ 

#ER0971 500 u 

#ER0975 1000 u 

#ER0972 2500 u 





Concentration 

10 u/μl 




NheI is supplied in: 

 


 

NlaIII Thermo Scientific enzymes FastDigest NlaIII and Hin1II 

NlaIV Thermo Scientific enzymes FastDigest NlaIV and BspLI 

20’ 



100 20-50 0-20 0-20 100 0-20 








Note 

 

10-fold more NheI for complete digestion 

 

 


132 

NmuCI 

5’...G T S A C ...3’ 

3’... C A S T G ...5’ 

#ER1511 200 u 





FastDigest NmuCI 

NotI 

5’...G CG G C C G C...3’ 

3’...C G C C G G C G...5’ 

#ER0591 300 u 

#ER0595 500 u 

#ER0592 1500 u 

#ER0593 HC, 1500 u 





FastDigest NotI 


Concentration 

10 u/μl 




NmuCI is supplied in: 

 


 

20’ 

Concentration 

10 u/μl 

50 u/μl, HC 




NotI is supplied in: 

 

0.1 mM EDTA, 1 mM DTT, 0.02% Triton X-100, 

 

NruI Thermo Scientific enzymes FastDigest NruI and Bsp68I 

NsbI 

5’...T G CG C A...3’ 

3’...A C G C G T...5’ 

#ER1221 400 u 





Concentration 

10 u/μl 




NsbI is supplied in: 

 


 

NsiI Thermo Scientific enzymes FastDigest Nsil and Mph1103I 

NspI Thermo Scientific enzymes FastDigest NspI and XceI 

NspV Thermo Scientific enzymes FastDigest Bsp119I and Bsp119I 

20’ 



0-20 20-50 50-100 100 20-50 50-100 




 




0-20 0-20 100 20-50 0-20 20-50 








Note 

 

5-fold more NotI for complete digestion than 

linear DNAs. 



20-50 50-100 0-20 20-50 100 20-50 







DNA in 16 hours.

OliI 

5’...C A C N NN N G T G...3’ 

3’...G T G N N N N C A C...5’ 

#ER1631 200 u 

#ER1632 1000 u 





FastDigest AleI 

PacI 

5’...T T A A TT A A...3’ 

3’...A A T T A A T T...5’ 

#ER2201 250 u 

#ER2202 1250 u 


10X Buffer PacI 1 ml 


FastDigest PacI 

PaeI 

5’...G C A T GC...3’ 

3’...C G T A C G...5’ 

#ER0601 500 u 

#ER0602 2500 u 





FastDigest SphI 

Concentration 

10 u/μl 

20’ 




OliI is supplied in: 

 


 

Concentration 

10 u/μl 

20’ 




PaeI is supplied in: 

 

50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 

0.15% Triton X-100, 0.5 mg/ml BSA 

 

PaeR7I Thermo Scientific enzymes FastDigest XhoI and XhoI 

20’ 

Concentration 

10 u/μl 


1X Buffer PacI at 37°C. 


PacI is supplied in: 

 


0.2 mg/ml BSA and 50% glycerol. 



0-20 0-20 0-20 100 0-20 50-100 




 

EcoKI, EcoBI: may overlap– blocked 







20-50 20-50 0-20 0-20 0-20 0-20 






for complete digestion of 1 μg of agarose- 

 

recognition sequence in 16 hours. 



100 50-100 0-20 0-20 50-100 0-20 









134 

PagI 

5’...TC A T G A...3’ 

3’...A G T A C T...5’ 

#ER1281 400 u 

#ER1282 2000 u 




FastDigest BspHI 

PasI 

5’...C CC W G G G...3’ 

3’...G G G W C C C...5’ 

#ER1861 200 u 


10X Buffer PasI 1 ml 

PauI 

5’...GC G C G C...3’ 

3’...C G C G C G...5’ 

#ER1091 200 u 

#ER1092 1000 u 





FastDigest BssHII 


Concentration 

10 u/μl 




PagI is supplied in: 

 


 

Concentration 

10 u/μl 


1X Buffer PasI at 55°C. 


PasI is supplied in: 

 


 

Concentration 

10 u/μl 




PauI is supplied in: 

 


 



0-20 50-100 100 NR NR NR 


Dam, EcoBI: may overlap – cleavage impaired 

 






Note 

dam 





NR NR NR NR NR NR 







Note 

 

 

results in star activity. 



0-20 0-20 100 100 0-20 100 







DNA in 16 hours.

PciI Thermo Scientific enzyme PscI 

PdiI 

5’...G C CG G C...3’ 

3’...C G G C C G...5’ 

#ER1521 200 u 

#ER1522 1000 u 





PdmI 

5’...G A A N NN N T T C...3’ 

3’...C T T N N N N A A G...5’ 

#ER1531 500 u 

#ER1532 2500 u 




FastDigest PdmI 

PfeI 

5’...GA W T C...3’ 

3’...C T W A G...5’ 

#ER1781 500 u 





FastDigest TfiI 

Concentration 

10 u/μl 

20’ 




PdiI is supplied in: 

 

1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA, 

 

20’ 

Concentration 

10 u/μl 




PdmI is supplied in: 

 

1 mM DTT, 5 mM MgCl2, 0.2 mg/ml BSA 

 

Concentration 

10 u/μl 

20’ 




PfeI is supplied in: 

 

1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA 

 



50-100 20-50 0-20 0-20 100 50-100 







pBR322 DNA in 16 hours. 

Note 

 


 

with PdiI, the same as with its prototype NaeI. 



20-50 50-100 0-20 0-20 100 0-20 











0-20 20-50 100 50-100 20-50 50-100 






136 

Pfl23II 

5’...CG T A C G...3’ 

3’...G C A T G C...5’ 

#ER0851 300 u 




FastDigest BsiWI 

PflFI Thermo Scientific enzymes FastDigest PsyI and PsyI 

PflMI Thermo Scientific enzymes FastDigest PflMI and Van91I 

PfoI 

5’...TC C N G G A...3’ 

3’...A G G N C C T...5’ 

#ER1751 200 u 




FastDigest PfoI 


Concentration 

3 u/μl 




Pfl23II is supplied in: 

 


 

Concentration 

10 u/μl 




PfoI is supplied in: 

 


 

PhoI Thermo Scientific enzymes FastDigest HaeIII and BsuRI 

PinAI Thermo Scientific enzymes FastDigest AgeI and BshTI 

PleI Thermo Scientific enzymes FastDigest MlyI and SchI 

PmaCI Thermo Scientific enzymes FastDigest PmlI and Eco72I 

PmeI Thermo Scientific enzymes FastDigest MssI and MssI 

PmlI Thermo Scientific enzymes FastDigest PmlI and Eco72I 

20’ 

20’ 



20-50 50-100 20-50 20-50 100 0-20 










0-20 20-50 50-100 0-20 100 50-100 



 


 

EcoKI, EcoBI – no effect. 





Note 

 

dam methylation and blocked by overlapping 

dcm methylation. To avoid dam or dcm 


as GM2163.

Ppu21I 

5’...Y A CG T R...3’ 

3’...R T G C A Y...5’ 

#ER1971 500 u 


10X Buffer Ppu21I 1 ml 



PpuMI Thermo Scientific enzymes FastDigest PpuMI and Psp5II 

PscI 

5’...AC A T G T...3’ 

3’...T G T A C A...5’ 

#ER1871 200 u 

#ER1872 1000 u 



Concentration 

10 u/μl 


1X Buffer Ppu21I at 30°C. 


Ppu21I is supplied in: 

 


 

20’ 

Concentration 

10 u/μl 




PscI is supplied in: 

 


 

PshAI Thermo Scientific enzymes FastDigest PshAI and BoxI 

PshBI Thermo Scientific enzymes FastDigest AseI and VspI 

PsiI Thermo Scientific enzymes FastDigest PsiI and AanI 

20’ 



50-100* 100* 20-50 NR NR NR 

* 









Note 

 

activity. 



20-50 20-50 0-20 0-20 100 0-20 








Note 

 

PciI, is inhibited by nonionic detergents 

 

 


138 

Psp5II 

5’...R GG W C C Y...3’ 

3’...Y C C W G G R...5’ 

#ER0761 500 u 





FastDigest PpuMI 

Psp1406I 

5’...A AC G T T...3’ 

3’...T T G C A A...5’ 

#ER0941 300 u 

#ER0942 1500 u 






Concentration 

10 u/μl 




Psp5II is supplied in: 

 


 

Concentration 

10 u/μl 

20’ 




Psp1406I is supplied in: 

 

1 mM DTT, 1 mM EDTA, 

 



0-20 100 20-50 20-50 50-100 100 









Note 

dcm 

methylation. To avoid dcm methylation, 




100 50-100 0-20 20-50 100 0-20 









PspGI Thermo Scientific enzymes EcoRII, 

FastDigest MvaI and MvaI 

PspOMI Thermo Scientific enzymes FastDigest ApaI and ApaI 

FastDigest Bsp120I and Bsp120I 

PstI 

5’...C T G C AG...3’ 

3’...G A C G T C...5’ 

#ER0611 


3000 u 



#ER0615 


10,000 u 



#ER0612 


5x3000 u 




FastDigest PstI 

Concentration 

10 u/μl 




PstI is supplied in: 

 


 


Dam, Dcm,CpG, EcoKI, EcoBI – no effect. 



50-100 50-100 100 100 50-100 50-100 





Note 

 

adjacent runs of G-C base pairs confers 

significant resistance to cleavage 

 

 

 

methylated by AluI methyltransferase.

PsuI 

5’...RG A T C Y...3’ 

3’...Y C T A G R...5’ 

#ER1551 500 u 





FastDigest PsuI 

PsyI 

5’...G A C NN N G T C...3’ 

3’...C T G N N N C A G...5’ 

#ER1331 1000 u 





FastDigest PsyI 

Concentration 

10 u/μl 



Concentration 

10 u/μl 




PsyI is supplied in: 

 


 

PteI Thermo Scientific enzyme FastDigest BssHII 

PvuI 

5’...C G A TC G...3’ 

3’...G C T A G C...5’ 

#ER0621 300 u 

#ER0622 1500 u 






Concentration 

10 u/μl 




PvuI is supplied in: 

 

0.1 mM EDTA, 1 mM DTT, 0.2 mg/ml BSA and 

 



100 20-50 0-20 0-20 50-100 0-20 


PsuI is supplied in: 

 


 






100 50-100 0-20 0-20 50-100 0-20 










0-20 20-50 50-100 100 50-100 100 









140 

PvuII 

5’...C A GC T G...3’ 

3’...G T C G A C...5’ 

#ER0631 


2500 u 



#ER0635 5000 u 

#ER0633 HC, 12500 u 





FastDigest PvuII 

5’...G TA C...3’ 

3’...C A T G...5’ 

#ER1121 


1000 u 


#ER1122 


5000 u 



FastDigest RsaI 


Concentration 

10 u/μl 

50 u/μl, HC 




PvuII is supplied in: 

 


 

RcaI Thermo Scientific enzymes FastDigest BspHI and PagI 

RruI Thermo Scientific enzyme FastDigest NruI 

RsaI 

RseI 

5’...C A Y N NN N R T G...3’ 

3’...G T R N N N N Y A C...5’ 

#ER2001 200 u 

#ER2002 1000 u 





FastDigest MslI 

Concentration 

10 u/μl 



Concentration 

10 u/μl 




RseI is supplied in: 

 


 

RsrII Thermo Scientific enzymes FastDigest RsrII and CpoI 

20’ 



50-100* 100 20-50 50-100 20-50* 20-50* 

* 








Note 

 

PvuII may result in star activity. 



50-100 20-50 0-20 0-20 100 0-20 


RsaI is supplied in: 

 


 




 



0-20 50-100 50-100 100 20-50 100 








DNA in 16 hours.

SacI 

5’...G A G C TC...3’ 

3’...C T C G A G...5’ 

#ER1131 1200 u 

#ER1135 


2000 u 

10X Buffer SacI 1 ml 


#ER1132 


5x1200 u 

10X Buffer SacI 2x1 ml 




SacII Thermo Scientific enzyme Cfr42I 

SalI 

5’...GT C G A C...3’ 

3’...C A G C T G...5’ 

#ER0641 1500 u 

#ER0645 


2000 u 



#ER0642 


5x1500 u 




FastDigest SalI 

20’ 

Concentration 

10 u/μl 


1X Buffer SacI at 37°C. 


SacI is supplied in: 

 


 







Concentration 

10 u/μl 




SalI is supplied in: 

 


 

SanDI Thermo Scientific enzyme FastDigest SanDI 

SapI Thermo Scientific enzymes FastDigest SapI and LguI 

SaqAI Thermo Scientific enzyme FastDigest MseI 

20’ 



50-100 20-50 0-20 0-20 50-100 20-50 

Note 

 

GAGmCTC but not GAGCTmC and insensitive 

to adenine methylation at GmAGCTC. 

 

to block SacI. 

 

5-fold more SacI for complete digestion than 

linear DNA. 



0-20 0-20 100 20-50 0-20 50-100 








Note 

 

require 10-fold overdigestion with SalI to 

achieve complete digestion. 

 


142 

SatI 

5’...G CN G C...3’ 

3’...C G N C G...5’ 

#ER1641 200 u 

#ER1642 1000 u 





FastDigest Fnu4HI 

SauI Thermo Scientific enzymes FastDigest Bsu36I and Eco81I 

Sau96I Thermo Scientific enzymes FastDigest Sau96I and Cfr13I 

Sau3AI Thermo Scientific enzymes FastDigest Sau3AI and Bsp143I ; 


FastDigest MboI and MboI 

SbfI Thermo Scientific enzymes FastDigest SbfI and SdaI 

ScaI 

5’...A G TA C T...3’ 

3’...T C A T G A...5’ 

#ER0431 


1000 u 

10X Buffer ScaI 1 ml 

#ER0432 


5000 u 

10X Buffer ScaI 2x1 ml 


FastDigest ScaI 


Concentration 

10 u/μl 

20’ 




SatI is supplied in: 

 


 

Concentration 

10 u/μl 


1X Buffer ScaI at 37°C. 


ScaI is supplied in: 

 


 



20-50 100 20-50 20-50 50-100 20-50 




Note 

 




0-20 0-20 0-20 0-20 0-20 0-20 








Note 

 

20-fold more ScaI for complete digestion 

than linear DNAs.

SchI 

5’...G A G T C(N) 5...3’ 

3’...C T C A G(N) 5 ...5’ 

#ER1371 1000 u 




FastDigest MlyI 

Concentration 

10 u/μl 

20’ 




SchI is supplied in: 

 


 

ScrFI Thermo Scientific enzymes FastDigest ScrFI and Bme1390I 

SdaI 

5’...C C T G C AG G...3’ 

3’...G G A C G T C C...5’ 

#ER1191 300 u 

#ER1192 1500 u 


10X Buffer SdaI 1 ml 



FastDigest SbfI 

SduI 

5’...G D G C HC...3’ 

3’...C H C G D G...5’ 

#ER0651 500 u 


10X Buffer SduI 1 ml 



Concentration 

10 u/μl 


1X Buffer SdaI at 37°C. 


SdaI is supplied in: 

 


 

Concentration 

10 u/μl 


1X Buffer SduI at 37°C. 

SecI Thermo Scientific enzymes FastDigest BsaJI and BseDI 

SexAI Thermo Scientific enzyme FastDigest SexAI 

20’ 



20-50 50-100 0-20 0-20 100 0-20 


Dam, Dcm,CpG, EcoKI – no effect. 


Note 

 


 




 





NR NR 0-20 0-20 NR 20-50 







Note 

 




NR 50-100* 50-100 0-20 NR NR 

* 


SduI is supplied in: 

 


 





144 

SfaAI 

5’...G C G A TC G C...3’ 

3’...C G C T A G C G...5’ 

#ER2091 1000 u 






Concentration 

10 u/μl 




SfaAI is supplied in: 

 


 

SfaNI Thermo Scientific enzymes FastDigest SfaNI and LweI 

SfcI Thermo Scientific enzymes FastDigest SfcI and BfmI 

SfeI Thermo Scientific enzymes FastDigest SfcI and BfmI 

SfiI 

5’...G G C C N N N NN G G C C...3’ 

3’...C C G G N N N N N C C G G...5’ 

#ER1821 1000 u 





FastDigest SfiI 

Concentration 

10 u/μl 




SfiI is supplied in: 

 

10 mM MgCl2, 1 mM DTT, 0.15% Triton X-100, 

 




 

SfoI Thermo Scientific enzymes FastDigest EheI and EheI ; 

Thermo Scientific enzyme FastDigest SspDI 

SfuI Thermo Scientific enzymes FastDigest Bsp119I and Bsp119I 

SgfI Thermo Scientific enzymes FastDigest AsiSI and SfaAI 



50-100 0-20 0-20 0-20 100 0-20 







 

recognition site in 16 hours. 



50-100 100 20-50 0-20 100 0-20 





Note 

 

dcm 



 


The two sites can be on either the same 

 

 

Therefore also an oligonucleotide harboring 

a SfiI recognition site can be supplemented.

SgeI 

5’... m5C N N G (N) 9 ...3’ 

3’... G N N C (N) 13 ...5’* 

* SgeI cleaves DNA targets containing 5-methylcytosine on 

one or both DNA strands 

#ER2211 250 u 


10X Buffer SgeI 1 ml 

SgrDI 

5’...C GT C G A C G...3’ 

3’...G C A G C T G C...5’ 

#ER2031 200 u 




SgsI 

5’...G GC G C G C C...3’ 

3’...C C G C G C G G...5’ 

#ER1891 300 u 

#ER1892 1500 u 




FastDigest AscI 

20’ 

Concentration 

3 u/μl 


1X Buffer SgeI at 37°C. 


SgeI is supplied in: 

 

1 mM DTT, 1 mM EDTA and 50% glycerol. 



Dcm: always cleaves DNA methylated by Dcm 

methyltransferase 

CpG: cleaves targets overlapping with CpG 

methylated sequences 

Concentration 

10 u/μl 

20’ 

Concentration 

5 u/μl 




SgrDI is supplied in: 

 


 

20’ 




SgsI is supplied in: 

 


 



0-20 0-20 0-20 NR NR NR 


A minimum of 3 units of the enzyme is 

required for complete digestion of 1 μg of 

agarose-embedded lambda DNA in 16 hours. 

Note 

 

methyltransferases will be a substrate for SgeI. 

 

may result in nonspecific cleavage. 

 

are required for an efficient cleavage. 

 

digestion of methylated DNA depends on 

the number of SgeI recognition sites. DNA 

cleavage products generated by target site 

cleavage facilitate the nonspecific cleavage 

by SgeI. Therefore, optimization of enzyme 

amount is recommended for DNA cleavage. 

E.coli dcm + 

 

cleavage efficiency control. SgeI cleaves all 

six dcm-methylated targets on pBR322 DNA. 



0-20 0-20 0-20 100 NR 100 










0-20 0-20 0-20 50-100 100 50-100 









146 

SinI Thermo Scientific enzymes FastDigest AvaII and Eco47I 

SmaI 

5’...C C CG G G...3’ 

3’...G G G C C C...5’ 

#ER0661 1200 u 

#ER0665 


2000 u 


#ER0662 5x1200 u 

#ER0663 HC, 6000 u 




FastDigest SmaI 

SmiI 

5’...A T T TA A A T...3’ 

3’...T A A A T T T A...5’ 

#ER1241 1000 u 





FastDigest SwaI 

SmlI Thermo Scientific enzyme SmoI 


Concentration 

10 u/μl 

50 u/μl, HC 




SmaI is supplied in: 

 


 

Concentration 

10 u/μl 

20’ 

20’ 




SmiI is supplied in: 

 


 



50-100 0-20 0-20 0-20 100 0-20 








Note 

 

SmaI has a half-life of 15 min at 37°C. 

 



0-20 0-20 100 20-50 0-20 20-50 








Note

SmoI 

5’...CT Y R A G...3’ 

3’...G A R Y T C...5’ 

#ER1981 200 u 



Concentration 

10 u/μl 




SmoI is supplied in: 

 


 

SnaI Thermo Scientific enzymes FastDigest BstZ17I and Bst1107I 

SnaBI Thermo Scientific enzymes FastDigest SnaBI and Eco105I 

SpeI Thermo Scientific enzymes FastDigest SpeI and BcuI 

SphI Thermo Scientific enzymes FastDigest SphI and PaeI 

SplI Thermo Scientific enzymes FastDigest BsiWI and Pfl23II 

Sse232I Thermo Scientific enzymes FastDigest MreI and MreI 

Sse8387I Thermo Scientific enzymes FastDigest SbfI and SdaI 

SsiI 

5’...CC G C...3’ 

3’...G G C G...5’ 

#ER1791 200 u 






Concentration 

10 u/μl 

20’ 





50-100 20-50 0-20 20-50 100 20-50 



 







Note 

 

dam 





NR 20-50 100 50-100 NR 100 


SsiI is supplied in: 

 


 





148 

SspI 

5’...A A TA T T...3’ 

3’...T T A T A A...5’ 

#ER0771 500 u 

#ER0772 2500 u 





FastDigest SspI 


Concentration 

10 u/μl 




SspI is supplied in: 

 


 

SspBI Thermo Scientific enzymes FastDigest Bsp1407I and Bsp1407I 

SspDI 

5’...GG C G C C...3’ 

3’...C C G C G G...5’ 

#ER2191 250 u 



Concentration 

10 u/μl 




SspDI is supplied in: 

 


 

20’ 



20-50 100 0-20 50-100 100 20-50 







Note 

 




20-50 0-20 NR 0-20 100 20-50 








StuI Thermo Scientific enzymes FastDigest StuI and Eco147I 

StyI Thermo Scientific enzymes FastDigest StyI and Eco130I 

StyD4I Thermo Scientific enzymes FastDigest ScrFI and Bme1390I 

SwaI Thermo Scientific enzymes FastDigest SwaI and SmiI

TaaI 

5’...A C NG T...3’ 

3’...T G N C A...5’ 

#ER1361 200 u 

#ER1362 1000 u 




FastDigest TaaI 

Tail 

* Unlike MaeII, TaiI produces DNA 

fragments with a 4-base 3’-extension 

5’... A C G T...3’ 

3’... T G C A ...5’ 

#ER1141 400 u 

#ER1142 


2000 u 




1 ml 

TaqI 

5’...TC G A...3’ 

3’...A G C T...5’ 

#ER0671 


3000 u 

10X Buffer TaqI 2x1 ml 


#ER0672 


5x3000 u 

10X Buffer TaqI 4x1 ml 




Concentration 

10 u/μl 



To ensure higher efficiency of digestion, perform 

the cleavage reaction under paraffin oil. 


TaaI is supplied in: 

 


 

Concentration 

10 u/μl 






TaiI is supplied in: 

 


 

Concentration 

10 u/μl, 


1X Buffer TaqI at 65°C. 




TaqI is supplied in: 

 


 



0-20 0-20 0-20 50-100 100 100 




 


Note 

 



50-100 50-100 20-50 100 100 50-100 





Note 

 

10% activity. 




Note 

dam 


a dam – , dcm – Activity in Five Buffer System, % 


0-20 20-50 20-50 20-50 20-50 20-50 


 

 

at 37°C. 

 

 

 


150 

TasI 

5’...A A T T ...3’ 

3’... T T A A ...5’ 

#ER1351 


1000 u 



#ER1352 


5000 u 




FastDigest Tsp509I 

TatI 

5’...WG T A C W...3’ 

3’...W C A T G W...5’ 

#ER1291 100 u 




FastDigest TatI 

TauI 

5’...G C S GC...3’ 

3’...C G S C G...5’ 

#ER1651 50 u 

#ER1652 250 u 







Concentration 

10 u/μl 






TasI is supplied in: 

 


 

Concentration 

5 u/μl 






TatI is supplied in: 

 


 

Concentration 

3 u/μl 




TauI is supplied in: 

 


 

TfiI Thermo Scientific enzymes FastDigest TfiI and PfeI 

TliI Thermo Scientific enzymes FastDigest XhoI and XhoI 



100 50-100 20-50 0-20 20-50 0-20 




Note 

 

10% activity. 



NR 50-100* 20-50 20-50 100* 0-20 

* 




Note 

 

 




100 50-100 0-20 0-20 20-50 0-20 




Note 

 

 




 


presence of SDS prior to electrophoresis.

Tru1I 

5’...TT A A ...3’ 

3’...A A T T...5’ 

#ER0981 300 u 

#ER0982 1500 u 

#ER0983 HC, 1500 u 






Concentration 

10 u/μl 

50 u/μl, HC 






Tru1I is supplied in: 

 


 

Tru9I Thermo Scientific enzymes FastDigest Tru1I and Tru1I ; 

Thermo Scientific enzyme FastDigest MseI 

TscAI 

5’... N N C A S T G N N...3’ 

3’... N N G T S A C N N ...5’ 

#ER2101 1000 u 




FastDigest TspRI 

Concentration 

10 u/μl 






TscAI is supplied in: 

 


 

Tsp4CI Thermo Scientific enzymes FastDigest TaaI and TaaI 

Tsp45I Thermo Scientific enzymes FastDigest NmuCI and NmuCI 

Tsp509I Thermo Scientific enzymes FastDigest Tsp509I and TasI 

TspMI Thermo Scientific enzymes Cfr9I ; 

FastDigest SmaI and SmaI 

TspRI Thermo Scientific enzymes FastDigest TspRI and TscAI 



50-100 50-100 20-50 100 50-100 100 




Note 

 

 

at 37°C. 



50-100 50-100 20-50 20-50 100 20-50 




Note 

 

activity. 

 

9-base 3’-extension. To avoid atypical 

DNA band patterns, use 6X DNA Loading 

 




152 

Tth111I Thermo Scientific enzymes FastDigest PsyI and PsyI 

Van91I 

5’...C C A N N N NN T G G...3’ 

3’...G G T N N N N N A C C...5’ 

#ER0711 400 u 

#ER0712 2000 u 





FastDigest PflMI 

VpaK11BI Thermo Scientific enzymes FastDigest AvaII and Eco47I 

VspI 

5’...A TT A A T...3’ 

3’...T A A T T A...5’ 

#ER0911 


1000 u 



#ER0912 


5000 u 




FastDigest AseI 

XagI 

5’...C C T N NN N N A G G...3’ 

3’...G G A N N N N N T C C...5’ 

#ER1301 1000 u 





FastDigest EcoNI 


Concentration 

10 u/μl 




Van91I is supplied in: 

 


 

Concentration 

10 u/μl 

20’ 

20’ 




VspI is supplied in: 

 


 

Concentration 

10 u/μl 

20’ 




XagI is supplied in: 

 


 



0-20 50-100 50-100 100 20-50 50-100 








Note 

dcm 





0-20 50-100 100 20-50 100 100 










0-20 20-50 50-100 100 20-50 50-100 







DNA in 16 hours.

XapI 

5’...RA A T T Y...3’ 

3’...Y T T A A R...5’ 

#ER1381 500 u 




FastDigest XapI 

XbaI 

5’...T C T A G A...3’ 

3’...A G A T C T...5’ 

#ER0681 


1500 u 


#ER0685 3000 u 

#ER0682 5x1500 u 

#ER0683 


HC, 7500 u 



FastDigest XbaI 

XceI 

5’...R C A T GY...3’ 

3’...Y G T A C R...5’ 

#ER1471 500 u 

#ER1472 2500 u 





Concentration 

10 u/μl 




XapI is supplied in: 

 


 

20’ 

Concentration 

10 u/μl 

50 u/μl, HC 




XbaI is supplied in: 

 


 

Concentration 

10 u/μl 

20’ 





50-100 100 0-20 0-20 100 20-50 








Note 

Greater than 15-fold overdigestion with XapI 




50-100 50-100 20-50 0-20 100 50-100 








Note 

dam 





50-100 0-20 0-20 0-20 100 0-20 


XceI is supplied in: 

 


 




154 

XhoI 

5’...CT C G A G...3’ 

3’...G A G C T C...5’ 

#ER0691 


2000 u 



#ER0695 5000 u 

#ER0692 5x2000 u 

#ER0693 HC, 10,000 u 





FastDigest XhoI 


Concentration 

10 u/μl 

50 u/μl, HC 




XhoI is supplied in: 

 


 

XhoII Thermo Scientific enzymes FastDigest PsuI and PsuI 

XmaI Thermo Scientific enzymes Cfr9I ; 


XmaIII Thermo Scientific enzymes FastDigest EagI and Eco52I 

XmaCI Thermo Scientific enzymes Cfr9I ; 


XmaJI 

5’...CC T A G G...3’ 

3’...G G A T C C...5’ 

#ER1561 200 u 

#ER1562 1000 u 





XmiI 

5’...G TM K A C...3’ 

3’...C A K M T G...5’ 

#ER1481 400 u 

#ER1482 2000 u 






Concentration 

10 u/μl 




 

 


 

Concentration 

10 u/μl 

20’ 




XmiI is supplied in: 

 


 



0-20 50-100 50-100 100 20-50 100 




 





Note 

 

5-fold more XhoI for complete digestion than 

linear DNA. 



20-50 50-100 50-100 50-100 100 50-100 









100 0-20 0-20 0-20 50-100 20-50 







DNA in 16 hours.

XmnI Thermo Scientific enzymes FastDigest PdmI and PdmI 

XspI Thermo Scientific enzymes FastDigest BfaI and FspBI 

ZraI Thermo Scientific enzymes FastDigest AatII and AatII 

Nicking Enzymes 

Nb.Bpu10I 

5’...C C T N A G C...3’ 

3’...G G A N T C G...5’ 

#ER1681 1000 u 




Concentration 

5 u/μl 


1X Buffer R: 

2, 100 mM KCl, 0.1 mg/ml BSA. 

Incubate at 37°C. 


Nb.Bpu10I is supplied in: 

 

KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA, 

50% glycerol. 

Nicking and Cleavage 

 

 

 

results in

156 

Nt.Bpu10I 

5’...C CT N A G C...3’ 

3’...G G A N T C G...5’ 

#ER2011 1000 u 




Nb.Mva1269I 

5’...G A A T G C...3’ 

3’...C T T A C G...5’ 

#ER2051 1000 u 





20’ 

Concentration 

5 u/μl 


1X Buffer R: 

2, 100 mM KCl, 0.1 mg/ml BSA. 



Nt.Bpu10I is supplied in: 

 

KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 mg/ml BSA, 

50% glycerol. 

Nicking and Cleavage 

 

 

 

results in

Homing Enzyme 

I-SceI is a site-specific homing enzyme encoded by a mitochondrial intron of Saccharomyces cerevisiae 

that promote the first step in mobility of the intron at the DNA level. They recognize and cleave an intronless allele of their cognate gene to insert a copy of 

 

long, 14-40 base pairs sequences and are, therefore, extremely rare-cutting enzymes. They allow the introduction of a single or several double-strand 

breaks into complex genomes. This capability makes these enzymes powerful tools in high-resolution physical mapping, genome organization analysis, gene 

 

I-SceI 

5’...T A G G G A T A AC A G G G T A A T...3’ 

3’...A T C C C T A T T G T C C C A T T A...5’ 

#ER1771 250 u 




 

100mM Mg-acetate 1 ml 

References 

1. Colleaux, L., et al., Recognition and cleavage site of the 

intron-encoded omega transposase, Proc. Natl. 

Acad. Sci. U.S.A.,85, 6022-6026, 1988. 

2. Monteihet, C., et al., Purification and characterization of 

the in vitro activity of I-SceI, a novel and highly specific 

endonuclease encoded by a group I intron, Nucleic Acids 

Res., 18, 1407-1413, 1990. 

3. Dujon, B., Group I introns as mobile genetic elements: facts 

and mechanistic speculations – review, Gene, 82, 

91-114, 1989. 

4. 

the house in order, Nucleic Acids Res., 25, 

3379-3388, 1997. 

5. Chevalier, B.S., Stoddard, B.L., Homing 

endonucleases:structural and functional insight into the 

catalysis of intron/intein mobility, Nucleic Acids Res., 29, 

3757-3774, 2001. 

6. 

cutting endonucleases, Trends in Genetics, 12, 

224-228, 1996. 

20’ 

Concentration 

10 u/μl 


1X Buffer Tango : 

 

10 mM Mg-acetate, 66 mM K-acetate and 

0.1 mg/ml BSA. 



I-SceI is supplied in: 

 


50% glycerol. 



50-100 50-100 50-100 50-100 100 50-100 






see the 

 

Note 

 

defined recognition sequences. They can 

tolerate minor sequence changes, which 

only partially affect the cleavage reaction. 

 




 




 

Digestion of the Agarose-embedded DNA with I-SceI 

1. Immerse an agarose plug in 50-100 μl of the 1X Tango buffer without Mg-acetate* 

 

2. Add 20 u of the enzyme. 

3. Incubate 2 hours on ice. 

4. 

5. Incubate at 37°C for 1 hour. 

Note 

* Diffusion of the enzyme in the absence of Mg-acetate prior to digestion is necessary because I-SceI is unstable in the 

presence of Mg 2+ ions. 


158 


1.5. DNA digestion 

We recommend digesting 0.2-1.5 μg DNA with 

a 2-fold to 10-fold excess of enzyme in a total 

volume of 20 μl . A typical restriction enzyme 

digestion protocol is below. 

1. Add the following reaction components in 

the order indicated: 

Water, nuclease-free 16-16.5 μl 

10X recommended buffer for restriction enzyme 2 μl 

Substrate DNA 

Restriction enzyme 

Total volume 20 μl 

2. Mix gently and spin down briefly. 

3. Incubate at the optimal reaction 

temperature for 1-16 hours. 

Note 

 

 

the stated activity. In these cases, add the required 

additive and adjust the volume of water appropriately. 

See Table 1.5 and Table 1.6 for restriction enzyme buffer 

composition and reaction conditions, respectively. 

1.6. Digestion of PCR products 

The most convenient option for digestion 

of PCR-amplified DNA is the addition of a 

restriction enzyme directly to the reaction tube 

after completion of PCR. The majority of Thermo 

Scientific restriction enzymes are active in PCR 

buffers. 

However, digestion of PCR products in the 

amplification mixture is often inefficient. 

Therefore, PCR reaction mixture should not 

make more than 1/3 volume of digestion 

reaction mixture to avoid inhibitory effects. 

1. Add the following reaction components in 

the order indicated: 

 

PCR reaction mixture 

 

Water, nuclease-free 16-17 μl 

10X recommended buffer for restriction enzyme 2 μl * 

Restriction enzyme 


* Only 2 μl of 10X reaction buffer is required for unpurified PCR product 

in a 30 μl reaction volume. 

2. Mix gently and spin down briefly. 

3. Incubate at the optimal reaction 

temperature for 1-16 hours. 

Note 

For cloning applications, purification of PCR products prior 

to digestion is necessary to remove the active thermophilic 

DNA polymerase present in the PCR mixture. DNA 

polymerases may alter the ends of the cleaved DNA and 

reduce the yield of ligation. 

If the restriction enzyme requires special additives 

 

If cleavage of the PCR product is inefficient, purify the PCR 

 

prior to digestion. 

After digestion, gel-purify the PCR product with the 

 

 

which compete with the insert in a ligation reaction. 


1.7. Setting up double digestion 

1.7.1. Double digestion with 

FastDigest enzymes 

For protocols and recommendations for 

FastDigest restriction enzymes see p.72. 

1.7.2. DoubleDigest Engine 

DoubleDigest engine 

www.thermoscientific.com/doubledigest 

a web tool for finding information on buffer 

and reaction conditions for double digests. 

Simply select two restriction enzymes required 

for digestion, submit the query and follow the 

recommendations. 

1.7.3. Double digestion 

in the universal Tango Buffer 

 

conditions for your double digestion reactions in 

the universal Tango buffer. 

1. Determine the concentration of Tango buffer 

recommended for each restriction enzyme. 

2. If the recommended concentration of Tango 

buffer is the same for both enzymes, the 

digests can be performed at the same time. 

3. If the two restriction enzymes require 

different concentrations of Tango buffer, 

perform the digestions sequentially. Set-up 

 

buffer, then add an 

additional aliquot of the 10X Tango buffer 

 

2X Tango buffer concentration to optimize 

conditions for the second enzyme. 

Note 

If both 1X and the 2X concentrations of Tango buffer are 

suitable for the double digest, use the 2X concentrated buffer 

to reduce the probability of star activity. 

1.7.4. Double digestion in the Five 

Buffer System 

 

Thermo Scientific restriction enzymes in the 

Five Buffer System. 

1. Determine which color-coded buffers are 

recommended for each enzyme. 

2. If possible, use the buffer in which both 

enzymes have 100% activity. 

3. If this is not possible, choose the buffer in 

which both enzymes maintain at least 20% 

of their activity. Increase the amount of the 

enzymes in your digest according to their 

activity in that buffer. 

Note 

For enzymes that are prone to relaxation of target specificity, 

use a buffer in which they do not exhibit star activity. 

1.7.5. Digestion with enzymes having 

different optimum temperatures 

For double digestion with enzymes working at 

different temperatures, perform sequential DNA 

cleavage. The optimal reaction temperature 

for each restriction enzyme is indicated both 

in the product description and the Certificate 

of Analysis. Information about the activity of 

mesophilic and thermophilic restriction enzymes 

at 37°C is given in Table 1.7 on p.165. 

1.7.6. Sequential digestion 

If double digestion is not possible due to buffer 

incompatibility or star activity, perform sequential 

digestions in the optimal buffer for each enzyme. 

1. Digest the DNA with the first restriction 

enzyme in its optimal buffer. 

2. 

 

extraction and ethanol precipitation. 

3. Digest the DNA with the second restriction 

enzyme in its optimal buffer. 

1.8. Stability during prolonged 

incubation 

The stability of restriction enzymes in a reaction 

mixture depends on the nature of the enzyme, 

the buffer composition and the incubation 

temperature. 

If a restriction enzyme retains its activity in 

the reaction mixture for more than one hour, 

DNA can be digested with less enzyme in 

a prolonged incubation period. The exact 

quantities of enzymes sufficient for overnight 

digestion are listed in the Table 1.6 on p.161. 

1.9. Dilution of restriction 

enzymes 

 

is available for applications that require diluted 

enzymes. 

Enzymes diluted in this buffer retain 50-100% 

activity after storage for one month at -20°C. 

1.10. Partial digestion of DNA 

Certain cloning experiments may require 

incomplete DNA cleavage. This partial digestion 

of the DNA can be achieved by using the 

following conditions: 

 

enzyme in the reaction mixture, 

 

 

For certain targets, partial cleavage of the 

desired DNA site is inefficient due to site 

see Site

1.11. Digestion of 

agarose-embedded DNA 

1. Embed the substrate DNA in 1% low melting 

temperature agarose, 1 μg / 30 μl. 

2. 

GelSyringe or similar agarose dispensing 

system. 

3. Equilibrate the plug in 100 μl of the 

appropriate 1X restriction enzyme buffer for 

15 min. 

4. Place the plug in 100 μl of fresh 1X buffer 

see Table 

1.9 on p.168 for recommended quantities 

 

5. Incubate at 30-37°C for mesophilic 

enzymes or at 50-55°C for thermophilic 

enzymes for 4-16 hours. 

1.12. Inactivation of restriction 

enzymes 

Inactivation of restriction enzymes following 

a digestion reaction is often required for 

downstream applications. 

Thermal inactivation is a convenient method 

used to terminate enzyme activity. The majority 

of restriction enzymes can be heat-inactivated 

at 65°C or 80°C in 20 min. Information on the 

susceptibility of Thermo Scientific restriction 

enzymes to thermal inactivation is listed in the 

 

descriptions and Certificates of Analysis. 

All known restriction enzymes with exception 

of BmuI and BmrI require Mg 2+ for DNA 

 

 

is an alternative method that can be used to 

halt digestion. However, EDTA is generally not 

compatible with most downstream applications, 

therefore purification of the digested DNA 

 

see 

recommended. 


160 


Reaction Buffers 

Our Five Buffer System ensures the optimum 

reaction conditions for each restriction 

 

 

 

enzymes are supplied in color-coded tubes 

to indicate the recommended reaction buffer. 

The recommended buffer and/or the universal 

Tango buffer, which has been designed for 

double digestions of DNA, are supplied with 

each enzyme. Restriction enzyme buffers are 

also available separately. see Table 1.5 below 

for ordering information. For more information 

on double digestion see p.158 or visit the 

Thermo Scientific DoubleDigest engine 

at www.thermoscientific.com/doubledigest. 

Table 1.5. Reaction buffers for restriction enzymes. 

10X buffer 


Cat. 

# 

Quantity, 

ml 

Tris- 

HCl, 

mM 

Trisacetate, 

mM 

To ensure consistent enzyme performance, 

Thermo Scientific restriction enzyme buffers 

contain BSA, which enhances the stability of 

many enzymes and binds contaminants that 

may be present in DNA preparations. Multiple 

freeze-thaw cycles of the buffers will not cause 

BSA precipitation. 

Thermo Scientific restriction enzymes 

exhibit 100% of their certified activity in 

the recommended buffer. However, some 

enzymes require additives to achieve 100% 

activity. For example, AjuI, AlfI, BplI, BseMII 

 

S-adenosylmethionine, which is supplied with 

 

 

 

Bis-Tris 

MgCl 

Propane-HCl, 

2, 

mM 

mM 

NaCl, 

mM 

1X buffer composition 

KCl, 

mM 

Mgacetate, 

mM 

K-acetate, 

mM 

The following enzymes require unique buffers 

 

 

 

 

 

 

SgeI and TaqI. The compositions of these unique 

buffers are listed in Table 1.5 as well as in of 

restriction enzymes Certificates of Analysis. 

All Thermo Scientific restriction enzyme buffers 

should be stored at -20°C. 

Sodium 

glutamate, 

mM 

Triton 

EDTA, DTT, 

X-100, 

mM mM 

% 

10X Buffer B BB5 5x1 10 

Five Buffer System 

10 0.1 7.5 

10X Buffer G BG5 5x1 10 10 50 0.1 7.5 

10X Buffer O BO5 5x1 50 10 100 0.1 7.5 

10X Buffer R BR5 5x1 10 10 100 0.1 8.5 

10X Buffer Tango BY5 5x1 33 10 66 0.1 7.9 

Buffer Set for 


B30 

1 ml of each 

buffer 

Unique buffers 

10X Buffer AarI, AjiI, 

Bpu10I, ScaI, PasI 

B27 1 10 10 100 0.1 6.5 

10X Buffer BamHI, 

Lsp1109I, SgeI 

B57 5x1 10 5 100 0.02 0.1 8.0 

10X Buffer BfuI B59 1 50 15 100 0.1 7.9 

10X Buffer BseXI B31 1 50 2 100 0.1 7.5 

10X Buffer Bsp143I B13 1 33 10 66 0.02 0.1 7.9 

10X Buffer Cfr9I B02 1 10 5 200 0.1 7.2 

10X Buffer Cfr10I B04 1 10 5 100 0.02 0.1 8.0 

10X Buffer Eam1105I B25 1 10 5 100 0.1 7.5 

10X Buffer Ecl136II, PacI, 

SacI 

B26 1 10 10 0.1 6.5 

10X Buffer Eco52I B22 1 10 3 100 0.1 8.5 

10X Buffer EcoRI B12 5x1 50 10 100 0.02 0.1 7.5 

10X Buffer KpnI B29 1 10 10 0.02 0.1 7.5 

10X Buffer SdaI B24 1 37 15 150 0.1 7.0 

10X Buffer SduI, Ppu21I B23 1 10 3 150 0.1 7.2 

10X Buffer TaqI B28 1 10 5 100 0.1 8.0 

Dilution Buffer for 


B19 5x1 ml 10 100 1 1 50 0.2 7.4 

Gly 

cerol, 

% 

BSA, 

mg/ 

ml 

pH, 

at 

37°C

Recommended Reaction Conditions 

Table 1.6. Reaction conditions for restriction enzymes. 

Enzyme 

Units for 

overnight 

incubation, 

u/μg DNA 

Thermal 

inactivation 

in 20 min 

Recom- 

mended buffer 

for 100% 

activity 

B (blue) 

1X 

Enzyme activity in Thermo Scientific buffers, % 

G (green) 

1X 

O (orange) 

1X 

R (red) 

1X 

Tango (yellow) 

1X 2X 

Tango buffer 

for double 

digestion 

AanI 0.5 65° C Tango 50-100 50-100 0-20 0-20 100 20-50 1X or 2X 

AarI 1.0 65° C 

Unique 

 

NR NR 

0-20 

 

0-20 

 

NR 

50-100 

 

2X 

 

AatII 0.3 65° C Tango 50-100 20-50 0-20 0-20 100 20-50 1X or 2X 

Acc65I 0.3 65° C O 0-20 20-50 100 20-50 20-50 50-100 1X or 2X 

AdeI 0.5 NO G 0-20 100 20-50 100 100* 20-50 1X* or 2X 

AjiI 0.5 65° C Unique NR NR 20-50* NR NR 20-50* 2X* 

AjuI 0.5 65° C 

AlfI 1.0 65° C 

R 

 

R 

 

0-20 

 

0-20 

 

50-100 

 

0-20 

 

20-50 

 

0-20 

 

100 

 

100 

 

50-100 

 

0-20 

 

50-100 

 

20-50 

 

1X or 2X 

 

2X 

 

AloI 0.1 65° C R 0-20 0-20 0-20 100 20-50 100 1X or 2X 

AluI 0.1 65° C Tango 50-100 0-20 0-20 0-20 100 20-50 1X or 2X 

Alw21I 0.1 65° C O 0-20 20-50 100 50-100 20-50 50-100 1X or 2X 

Alw26I 0.2 65° C Tango 50-100 100 0-20 0-20 100 100 1X or 2X 

Alw44I 0.1 65° C Tango 50-100 100 0-20 50-100 100 50-100 1X or 2X 

ApaI 0.2 65° C B 100 20-50 0-20 0-20 20-50 0-20 1X 

BamHI 0.5 Unique 20-50* 100 20-50 50-100* 100* 50-100 1X* or 2X 

BauI 0.2 65° C Tango 0-20 50-100 0-20 50-100 100 50-100 1X or 2X 

BclI 0.1 G 20-50 100 20-50 20-50 100* 100 1X* or 2X 

BcnI 0.2 65° C Tango 20-50 50-100 50-100 50-100 100 50-100 1X or 2X 

BcuI 0.5 NO Tango 50-100 50-100 0-20 20-50 100 0-20 1X 

BfmI 1.0 65° C Tango 0-20 50-100 0-20 0-20 100 20-50 1X or 2X 

BfuI 1.0 80° C Unique NR NR 0-20 0-20 NR 50-100* 2X* 

BglI 0.1 65° C O 0-20 50-100 100 100 0-20 100 2X 

BglII 0.1 NO O 0-20 20-50 100 50-100 0-20 100 2X 

Bme1390I 0.2 80° C O 20-50 50-100 100 50-100 50-100 50-100 1X or 2X 

BoxI 0.5 80° C Tango 0-20 0-20 0-20 20-50 100 20-50 1X or 2X 

BpiI 0.3 65° C G 20-50 100 50-100 50-100 50-100 50-100 1X or 2X 

BplI 0.3 65° C 

Tango 

 

0-20 

 

20-50 

 

0-20 

 

0-20 

 

100 

 

20-50 

 

1X 

 

Bpu10I 0.2 80° C Unique 0-20 20-50* 50-100* 100* 50-100* 100* 1X* or 2X* 

Bpu1102I 0.1 80° C Tango 50-100 50-100 20-50 20-50 100 20-50 1X or 2X 

BseDI 0.2 80° C Tango 50-100 20-50 0-20 0-20 100 50-100 1X or 2X 

BseGI 0.2 80° C Tango 20-50 50-100 20-50 20-50 100 20-50 1X or 2X 

BseJI 0.1 NO O NR 100* 100 NR NR 100* 2X* 

BseLI 0.1 NO Tango 20-50 100 50-100 20-50 100 50-100 1X or 2X 

BseMI 0.3 80° C R 0-20 20-50 0-20 100 50-100 50-100 1X or 2X 

BseMII 

 

0.5 80° C 

Tango 

 

50-100 

 

50-100 

 

50-100 

 

50-100 

 

100 

 

50-100 

 

1X or 2X 

 

BseNI 0.1 80° C B 100 20-50 0-20 0-20 50-100 20-50 1X or 2X 

BseSI 0.1 G 20-50 100 0-20 20-50 50-100 0-20 1X 

BseXI 0.3 80° C Unique NR NR NR NR NR NR NR 

Bsh1236I 0.1 65°C R 0-20 0-20 50-100 100 20-50 50-100 1X or 2X 

Bsh1285I 0.2 80° C G 20-50 100 20-50 0-20 0-20 20-50 2X 

BshNI 0.3 65° C O 0-20 20-50 100 50-100 0-20 100 2X 

BshTI 0.1 80° C O 0-20 20-50 100 50-100 20-50 20-50 1X or 2X 

 


162 


Enzyme 


Units for 

overnight 

incubation, 

u/μg DNA 

Thermal 

inactivation 

in 20 min 

Recom- 

mended buffer 

for 100% 

activity 

B (blue) 

1X 


G (green) 

1X 

O (orange) 

1X 

R (red) 

1X 


1X 2X 

Tango buffer 

for double 

digestion 

Bsp68I 0.2 65° C O 0-20 20-50 100 50-100 20-50 50-100 1X or 2X 

Bsp119I 0.1 80° C Tango 20-50 0-20 0-20 0-20 100 100 1X or 2X 

Bsp120I 0.1 80° C B 100 20-50 0-20 20-50 50-100 0-20 1X 

Bsp143I 0.1 65° C Unique 20-50 20-50 0-20 0-20 50-100 20-50 1X or 2X 

Bsp1407I 0.1 65° C Tango 0-20 20-50 0-20 20-50 100 50-100 1X or 2X 

BspLI 0.3 65° C Tango 50-100 50-100 0-20 20-50 100 20-50 1X or 2X 

BspOI 0.2 80° C O 0-20 0-20 100 100 0-20 20-50 2X 

BspPI 0.5 80° C Tango 20-50 20-50 0-20 0-20 100 0-20 1X 

BspTI 0.1 65° C O 0-20 0-20 100 20-50 0-20 50-100 2X 

Bst1107I 0.1 80° C O 20-50 50-100 100 100 20-50 100 1X or 2X 

BstXI 0.1 80° C O 20-50 100 100 50-100 50-100 100 1X or 2X 

Bsu15I 0.1 65° C Tango 20-50 20-50 20-50 20-50 100 20-50 1X or 2X 

BsuRI 0.1 80° C R 20-50 20-50 50-100 100 50-100 100 1X or 2X 

BveI 0.2 65° C 

O 

 

0-20 

 

20-50 

 

100 

 

20-50 

 

50-100 

 

100 

 

1X or 2X 

 

CaiI 0.2 65° C Tango 20-50 20-50 20-50 50-100 100 50-100 1X or 2X 

Cfr9I 0.2 65° C Unique 0-20 0-20 0-20 0-20 20-50 0-20 1X 

Cfr10I 0.1 NO Unique 0-20 20-50 20-50 50-100* 20-50 50-100 1X or 2X 

Cfr13I 0.3 65° C Tango 50-100 50-100 20-50 20-50 100 20-50 1X or 2X 

Cfr42I 0.1 65° C B 100 50-100 0-20 0-20 50-100 0-20 1X 

CpoI 0.5 65° C Tango 20-50 50-100 50-100 20-50 100 50-100 1X or 2X 

CseI 0.5 R NR 50-100* 50-100 100 100* 50-100 1X* or 2X 

Csp6I 0.1 65° C B 100 50-100 0-20 0-20 50-100 0-20 1X 

DpnI 0.1 80° C Tango 100 100 50-100 50-100 100 50-100 1X or 2X 

DraI 0.1 65° C Tango 50-100 50-100 20-50 20-50 100 50-100 1X or 2X 

Eam1104I 0.5 65° C Tango 50-100 50-100 0-20 0-20 100 0-20 1X 

Eam1105I 0.1 65° C Unique 20-50 50-100 0-20 0-20 50-100 20-50 1X or 2X 

Ecl136II 0.2 65° C Unique 50-100 20-50 0-20 0-20 50-100 0-20 1X 

Eco24I 0.2 65° C Tango 50-100 50-100 0-20 20-50 100 0-20 1X 

Eco31I 0.3 65° C G 50-100 100 0-20 0-20 50-100 20-50 1X or 2X 

Eco32I 0.1 80° C R 0-20 50-100 50-100 100 20-50 100 1X or 2X 

Eco47I 0.3 65° C R 0-20 50-100 50-100 100 50-100 50-100 1X or 2X 

Eco47III 0.1 65° C O 0-20 20-50 100 100 50-100 100 1X or 2X 

Eco52I 0.2 65° C Unique 0-20 0-20 0-20 20-50 0-20 20-50 2X 

Eco57I 1.0 65° C 

G 

 

100 

 

100 

 

20-50 

 

20-50 

 

50-100 

 

50-100 

 

1X or 2X 

 

Eco72I 0.5 65° C Tango NR NR 0-20 0-20 100 20-50 1X or 2X 

Eco81I 0.1 80° C Tango 50-100 100 0-20 0-20 100 0-20 1X 

Eco88I 0.2 65° C Tango 100 50-100 0-20 0-20 100 20-50 1X or 2X 

Eco91I 0.1 65° C O 20-50 20-50 100 50-100 NR 100 2X 

Eco105I 0.5 65° C Tango 100* 50-100 0-20 0-20 100 0-20 1X 

Eco130I 0.2 65° C O 0-20 20-50 100 50-100 50-100 100 1X or 2X 

Eco147I 0.1 80° C B 100 50-100 20-50 20-50 50-100 0-20 1X 

EcoO109I 0.2 65° C Tango 50-100 20-50 20-50 20-50 100 100 1X or 2X 

EcoRI 0.2 65° C Unique 0-20 NR 100 100* NR 100 2X 

EcoRII 0.1 80° C O 20-50 50-100 100 50-100 20-50 50-100 1X or 2X 

EheI 1.0 65° C Tango 20-50 50-100 0-20 0-20 100 20-50 1X or 2X 

Esp3I 0.2 65° C 

Tango 

 

100 

 

20-50 

 

0-20 

 

0-20 

 

100 

 

0-20 

 

1X


Enzyme 

Units for 

overnight 

incubation, 

u/μg DNA 

Thermal 

inactivation 

in 20 min 

FaqI 1.0 80° C 

Recom- 

mended buffer 

for 100% 

activity 

Tango 

 

B (blue) 

1X 

20-50 

 


G (green) 

1X 

20-50 

 

O (orange) 

1X 

0-20 

 

R (red) 

1X 

0-20 

 


1X 2X 

100 

 

20-50 

 

Tango buffer 

for double 

digestion 

1X or 2X 

 

FspAI 0.2 65° C O 0-20 0-20 100 50-100 0-20 50-100 2X 

FspBI 0.3 65° C Tango 50-100 20-50 0-20 0-20 100 0-20 1X 

GsuI 1.0 65° C B 100 50-100 20-50 20-50 100 50-100 1X or 2X 

HhaI 0.1 NO Tango 50-100 50-100 20-50 20-50 100 20-50 1X or 2X 

Hin1I 0.1 65° C G 20-50 100 20-50 20-50 20-50 20-50 1X or 2X 

Hin1II 0.3 65° C G 50-100 100 20-50 50-100 50-100 50-100 1X or 2X 

Hin6I 0.1 65° C Tango 50-100 50-100 20-50 20-50 100 50-100 1X or 2X 

HincII 0.1 65° C Tango 50-100 50-100 20-50 50-100 100 50-100 1X or 2X 

HindIII 0.1 80° C R 0-20 20-50 0-20 100 50-100 50-100 1X or 2X 

HinfI 0.1 65° C R 0-20 20-50 50-100 100 50-100 50-100 1X or 2X 

HpaII 0.1 65° C Tango 50-100 50-100 0-20 20-50 100 20-50 1X or 2X 

HphI 0.1 65° C B 100 0-20 0-20 0-20 20-50 0-20 1X 

Hpy8I 0.1 80° C Tango 50-100 50-100 0-20 20-50 100 50-100 1X or 2X 

HpyF3I 0.2 65° C Tango 20-50 20-50 20-50 20-50 100 50-100 1X or 2X 

HpyF10VI 0.1 80° C Tango 0-20 0-20 0-20 0-20 100 50-100 1X or 2X 

KpnI 0.2 80° C Unique 20-50 0-20 0-20 0-20 20-50 0-20 1X 

Kpn2I 0.2 80° C Tango 50-100 50-100 0-20 20-50 100 50-100 1X or 2X 

KspAI 0.5 65° C B 100 50-100* 20-50 20-50 100* 50-100 1X* or 2X 

LguI 0.5 65° C Tango 20-50 50-100 20-50 20-50 100 20-50 1X or 2X 

Lsp1109I 0.5 65° C Unique 0-20 20-50* 50-100* 100* 20-50* 20-50* 1X* or 2X* 

LweI 0.2 65° C Tango 0-20 0-20 0-20 20-50 100 20-50 1X or 2X 

MauBI 0.1 65° C Tango 0-20 0-20 0-20 0-20 100 0-20 1X 

MbiI 0.2 65° C Tango 20-50 100 20-50 20-50 100 20-50 1X or 2X 

MboI 0.1 65° C R 50-100 50-100 50-100 100 50-100 100 1X or 2X 

MboII 1.0 65° C B 100 50-100 20-50 0-20 50-100 20-50 1X or 2X 

MlsI 0.5 65° C R 0-20 20-50 0-20 100 20-50 50-100 1X or 2X 

MluI 0.1 80° C R 0-20 20-50 50-100 100 20-50 50-100 1X or 2X 

MnlI 0.5 65° C G 50-100 100 20-50 20-50 20-50 20-50 1X or 2X 

Mph1103I 0.3 65° C R 0-20 50-100 20-50 100 50-100 50-100 1X or 2X 

MreI 0.5 80° C G 20-50 100 0-20 0-20 50-100 0-20 1X 

MspI 0.3 80° C Tango 50-100 50-100 0-20 0-20 100 50-100 1X or 2X 

MssI 0.5 65° C B 100 0-20 0-20 0-20 20-50 0-20 1X 

MunI 0.1 65° C G 100 100 0-20 0-20 100 0-20 1X 

MvaI 0.1 NO R 20-50 20-50 50-100 100 20-50* 100 1X* or 2X 

Mva1269I 0.1 65° C R 0-20 20-50 50-100 100 0-20 50-100 2X 

NcoI 0.1 65° C Tango 20-50 20-50 20-50 50-100 100 100 1X or 2X 

NdeI 0.2 65° C O 0-20 0-20 100 50-100 0-20 50-100 2X 

NheI 0.2 65° C Tango 100 20-50 0-20 0-20 100 0-20 1X 

NmuCI 0.1 65° C R 0-20 20-50 50-100 100 20-50 50-100 1X or 2X 

NotI 0.1 80° C O 0-20 0-20 100 20-50 0-20 20-50 2X 

NsbI 0.1 65° C Tango 20-50 50-100 0-20 20-50 100 20-50 1X or 2X 

OliI 0.2 65° C R 0-20 0-20 0-20 100 0-20 50-100 2X 

PacI 0.3 65° C Unique 20-50 20-50 0-20 0-20 0-20 0-20 NR 

PaeI 0.1 65° C B 100 50-100 0-20 0-20 50-100 0-20 1X 

PagI 0.2 80° C O 0-20 50-100 100 NR NR NR NR 

PasI 0.2 80° C Unique NR NR NR NR NR NR NR 

PauI 0.2 80° C R 0-20 0-20 100 100 0-20 100 2X 

 


164 


Enzyme 


Units for 

overnight 

incubation, 

u/μg DNA 

Thermal 

inactivation 

in 20 min 

Recom- 

mended buffer 

for 100% 

activity 

B (blue) 

1X 


G (green) 

1X 

O (orange) 

1X 

R (red) 

1X 


1X 2X 

Tango buffer 

for double 

digestion 

PdiI 0.5 65°C Tango 50-100 20-50 0-20 0-20 100 50-100 1X or 2X 

PdmI 0.5 65° C Tango 20-50 50-100 0-20 0-20 100 0-20 1X 

PfeI 0.1 65° C O 0-20 20-50 100 50-100 20-50 50-100 1X or 2X 

Pfl23II 0.3 65° C Tango 20-50 50-100 20-50 20-50 100 0-20 1X 

PfoI 0.1 65° C Tango 0-20 20-50 50-100 0-20 100 50-100 1X or 2X 

Ppu21I 0.5 65° C Unique 50-100* 100* 20-50 NR NR NR NR 

PscI 0.2 65° C Tango 20-50 20-50 0-20 0-20 100 0-20 1X 

Psp5II 0.2 80° C G 0-20 100 20-50 20-50 50-100 100 1X or 2X 

Psp1406I 0.5 65° C Tango 100 50-100 0-20 20-50 100 0-20 1X 

PstI 0.2 NO O 50-100 50-100 100 100 50-100 50-100 1X or 2X 

PsuI 0.5 80° C B 100 20-50 0-20 0-20 50-100 0-20 1X 

PsyI 0.1 80° C B 100 50-100 0-20 0-20 50-100 0-20 1X 

PvuI 0.2 R 0-20 20-50 50-100 100 50-100 100 1X or 2X 

PvuII 0.2 NO G 50-100* 100 20-50 50-100 20-50* 20-50* 1X* or 2X* 

RsaI 0.2 80° C Tango 50-100 20-50 0-20 0-20 100 0-20 1X 

RseI 0.5 65° C R 0-20 50-100 50-100 100 20-50 100 1X or 2X 

SacI 0.2 65° C Unique 50-100 20-50 0-20 0-20 50-100 20-50 1X or 2X 

SalI 0.1 65° C O 0-20 0-20 100 20-50 0-20 50-100 2X 

SatI 0.1 65° C G 20-50 100 20-50 20-50 50-100 20-50 1X or 2X 

ScaI 0.5 Unique 0-20 0-20 0-20 0-20 0-20 0-20 NR 

SchI 0.2 65° C Tango 20-50 50-100 0-20 0-20 100 0-20 1X 

SdaI 0.3 80° C Unique NR NR 0-20 0-20 NR 20-50 2X 

SduI 0.3 65° C Unique NR 50-100* 50-100 0-20 NR NR NR 

SfaAI 0.2 80° C Tango 50-100 0-20 0-20 0-20 100 0-20 1X 

SfiI 0.2 NO G 50-100 100 20-50 0-20 100 0-20 1X 

SgeI NR 65° C Unique 0-20 0-20 0-20 NR NR NR NR 

SgrDI 0.3 65° C R 0-20 0-20 0-20 100 NR 100 2X 

SgsI 0.1 65° C Tango 0-20 0-20 0-20 50-100 100 50-100 1X or 2X 

SmaI 0.2 65° C Tango 50-100 0-20 0-20 0-20 100 0-20 1X 

SmiI 0.1 65° C O 0-20 0-20 100 20-50 0-20 20-50 2X 

SmoI 0.2 80° C Tango 50-100 20-50 0-20 20-50 100 20-50 1X or 2X 

SsiI 0.5 65° C O NR 20-50 100 50-100 NR 100 2X 

SspI 0.1 65° C G 20-50 100 0-20 50-100 100 20-50 1X or 2X 

SspDI 0.1 80° C Tango 20-50 0-20 NR 0-20 100 20-50 1X or 2X 

TaaI 0.2 NO Tango 0-20 0-20 0-20 50-100 100 100 1X or 2X 

TaiI 0.3 NO R 50-100 50-100 20-50 100 100 50-100 1X or 2X 

TaqI 0.3 NO Unique 0-20 20-50 20-50 20-50 20-50 20-50 1X or 2X 

TasI 0.3 NO B 100 50-100 20-50 0-20 20-50 0-20 1X 

TatI 0.2 NO Tango NR 50-100* 20-50 20-50 100* 0-20 1X* 

TauI 1.0 NO B 100 50-100 0-20 0-20 20-50 0-20 1X 

Tru1I 0.2 NO R 50-100 50-100 20-50 100 50-100 100 1X or 2X 

TscAI 0.2 NO Tango 50-100 50-100 20-50 20-50 100 20-50 1X or 2X 

Van91I 0.1 65° C R 0-20 50-100 50-100 100 20-50 50-100 1X or 2X 

VspI 0.1 65° C O 0-20 50-100 100 20-50 100 100 1X or 2X 

XagI 0.1 65° C R 0-20 20-50 50-100 100 20-50 50-100 1X or 2X 

XapI 0.1 80° C Tango 50-100 100 0-20 0-20 100 20-50 1X or 2X 

XbaI 0.1 65°C Tango 50-100 50-100 20-50 0-20 100 50-100 1X or 2X 

XceI 0.2 65° C Tango 50-100 0-20 0-20 0-20 100 0-20 1X 

XhoI 0.1 80° C R 0-20 50-100 50-100 100 20-50 100 1X or 2X 

XmaJI 0.2 80° C Tango 20-50 50-100 50-100 50-100 100 50-100 1X or 2X


Enzyme 

Units for 

overnight 

incubation, 

u/μg DNA 

Thermal 

inactivation 

in 20 min 

Recom- 

mended buffer 

for 100% 

activity 

B (blue) 

1X 

* 

NR – buffer is not recommended, due to high star activity. 

NO – no thermal inactivation. 

 

Activity of Mesophilic and Thermophilic Enzymes at 37°C 

Table 1.7. Activity of mesophilic and thermophilic enzymes at 37°C. 

Enzyme 

Optimal 

temperature 

Activity at 

37°C, % 

Enzyme 

Optimal 

temperature 

Activity at 

37°C, % 

AloI 30 20 PasI 55 30 

BclI 55 50 Ppu21I 30

166 

Double Digestion in the Universal Tango Buffer 

Table 1.8. Double digestion in the universal Tango buffer. 

Thermo Scientific 

enzyme 


Recommended 

buffer 

Buffers for double digestions 

Tango 

1X 2X 

AanI Tango 

AarI AarI +oligo NR +oligo 

AatII Tango 

Acc65I O 

AdeI G * 

AjiI AjiI NR * 

AjuI R +SAM +SAM +SAM 

AlfI R +SAM NR +SAM 

AloI R 

AluI Tango 

Alw21I O 

Alw26I Tango 

Alw44I Tango 

ApaI B NR 

BamHI BamHI * 

BauI Tango 

BclI G * 

BcnI Tango 

BcuI Tango NR 

BfmI Tango 

BfuI BfuI NR * 

BglI O NR 

BglII O NR 

Bme1390I O 

BoxI Tango 

BpiI G 

BplI Tango +SAM +SAM NR 

Bpu10I Bpu10I * * 

Bpu1102I Tango 

BseDI Tango 

BseGI Tango 

BseJI O NR * 

BseLI Tango 

BseMI R 

BseMII Tango +SAM +SAM +SAM 

BseNI B 

BseSI G NR 

BseXI BseXI NR NR 

Bsh1236I R 

Bsh1285I G NR 

BshNI O NR 

BshTI O 

Bsp68I O 

Bsp119I Tango 

Bsp120I B NR 

Bsp143I Bsp143I 

Bsp1407I Tango 

BspLI Tango 

BspOI O NR 

BspPI Tango NR 

BspTI O NR 

Bst1107I O 

BstXI O 

Bsu15I Tango 


enzyme 

Recommended 

buffer 


Tango 

1X 2X 

BsuRI R 

BveI O +oligo +oligo +oligo 

CaiI Tango 

Cfr9I Cfr9I NR 

Cfr10I Cfr10I 

Cfr13I Tango 

Cfr42I B NR 

CpoI Tango 

CseI R * 

Csp6I B NR 

DpnI Tango 

DraI Tango 

Eam1104I Tango NR 

Eam1105I Eam1105I 

Ecl136II Ecl136II NR 

Eco24I Tango NR 

Eco31I G 

Eco32I R 

Eco47I R 

Eco47III O 

Eco52I Eco52I NR 

Eco57I G +SAM +SAM +SAM 

Eco72I Tango 


Eco88I Tango 

Eco91I O 


Eco130I O 

Eco147I B NR 

EcoO109I Tango 

EcoRI EcoRI NR 

EcoRII O 

EheI Tango 

Esp3I Tango +DTT +DTT NR 

FaqI Tango +SAM +SAM +SAM 

FspAI O NR 

FspBI Tango NR 

GsuI B 

HhaI Tango 

Hin1I G 

Hin1II G 

Hin6I Tango 

HincII Tango 

HindIII R 

HinfI R 

HpaII Tango 

HphI B NR 

Hpy8I Tango 

HpyF3I Tango 

HpyF10VI Tango 

KpnI KpnI NR 

Kpn2I Tango 

KspAI B * 

LguI Tango

Table 1.8. Double digestion in universal Tango buffer. 


enzyme 

Recommended 

buffer 


Tango 

1X 2X 

Lsp1109I Lsp1109I * * 

LweI Tango 

MauBI Tango NR 

MbiI Tango 

MboI R 

MboII B 

MlsI R 

MluI R 

MnlI G 

Mph1103I R 

MreI G NR 

MspI Tango 

MssI B NR 

MunI G NR 

MvaI R * 

Mva1269I R NR 

NcoI Tango 

NdeI O NR 

NheI Tango NR 

NmuCI R 

NotI O NR 

NsbI Tango 

OliI R NR 

PaeI B NR 

PacI PacI NR NR 

PagI O NR NR 

PasI PasI NR NR 

PauI R NR 

PdiI Tango 

PdmI Tango NR 

PfeI O 

Pfl23II Tango NR 

PfoI Tango 

Ppu21I Ppu21I NR NR 

PscI Tango NR 

Psp5II G 

Psp1406I Tango NR 

PstI O 

PsuI B NR 

PsyI B NR 

PvuI R 

PvuII G * * 

RsaI Tango NR 

RseI R 

SacI SacI 

SalI O NR 

SatI G 

ScaI ScaI NR NR 

SchI Tango NR 

SdaI SdaI NR 

SduI SduI NR NR 

SfaAI Tango NR 

SfiI G NR 

SgeI SgeI NR NR 

SgrDI R NR 

SgsI Tango 


enzyme 

Recommended 

buffer 


Tango 

1X 2X 

SmaI Tango NR 

SmiI O NR 

SmoI Tango 

SsiI O NR 

SspI G 

SspDI Tango 

TaaI Tango 

TaiI R 

TaqI TaqI 

TasI B NR 

TauI B NR 

TatI Tango * NR 

Tru1I R 

TscAI Tango 

Van91I R 

VspI O 

XagI R 

XapI Tango 

XbaI Tango 

XceI Tango NR 

XhoI R 

XmaJI Tango 

XmiI B 

I-SceI Tango 

Nb.Bpu10I R 

Nt.Bpu10I R NR 

Nb.Mva1269I O 

* – 

Optimal temperature for the enzymes listed is 37°C unless otherwise 

indicated in parenthesis. 

NR – buffer is not recommended because enzyme activity is less than 20% or 

star activity is too high. 

Cleavage efficiency 

20-50% 

50-100% 


168 

Digestion of Agarose-Embedded DNA 

Table 1.9. Digestion of agarose-embedded DNA. 


enzyme 


Units of enzyme 

required for DNA 

cleavage in 16 h 

AanI 5 

AarI 5 

AatII 5 

Acc65I 5 

AdeI 10 

AjiI 5 

AjuI 5 

AlfI 5 

Alw44I 5 

ApaI 5 

BamHI 5 

BauI 5 

BcuI 10 

BfuI 5 

BglI 5 

BglII 5 

BoxI 10 

BpiI 5 

BplI 10 

Bpu1102I 5 

BseSI 5 

Bsh1285I 5 

BshNI 5 

BshTI 20 

Bsp68I 5 

Bsp119I 5 

Bsp120I 5 

Bsp1407I 10 

BspOI 20 

BspTI 5 

Bst1107I 5 

BstXI 5 

Bsu15I 5 

CaiI 5 

Cfr9I 5 

Cfr10I 5 

Cfr42I 20 

DraI 5 

Eam1104I 5 

Eam1105I 5 

Ecl136II 5 

Eco24I 5 

Eco31I 5 

Eco32I 5 

Eco47III 5 

Eco52I 5 

Eco72I 5 

Eco81I 5 

Eco88I 5 

Eco91I 5 

Eco105I 5 

Eco130I 5 

Eco147I 5 

EcoO109I 5 

EcoRI 5 


enzyme 




EheI 20 

Esp3I 5 

FspBI 10 

Hin1I 5 

HindIII 5 

KpnI 5 

Kpn2I 5 

KspAI 5 

LguI 5 

MauBI 5 

MbiI 5 

MlsI 20 

MluI 5 

MssI 5 

MunI 5 

Mva1269I 5 

NcoI 5 

NdeI 5 

NheI 5 

NotI 5 

NsbI 5 

OliI 5 

PaeI 5 

PacI 5 

PagI 5 

PasI 10 

PauI 10 

PdiI 20 

PdmI 10 

Pfl23II 5 

PfoI 10 

Ppu21I 5 

PscI 5 

Psp5II 5 

Psp1406I 10 

PstI 5 

PsyI 5 

PvuI 5 

PvuII 5 

RseI 5 

SacI 5 

SalI 5 

ScaI 20 

SdaI 5 

SfaAI 5 

SfiI 5 

SgeI 3 

SgrDI 5 

SgsI 5 

SmaI 5 

SmiI 10 

SmoI 5 

SspI 5 

SspDI 5 

Van91I 5 


enzyme 




VspI 5 

XagI 5 

XapI 5 

XbaI 5 

XhoI 5 

XmaJI 10 

XmiI 5 

I-SceI 20 

Note 

For digestion of agarose-embedded DNA protocol 

see 1.11 on p.159.

Cleavage Efficiency Close to the Termini of PCR Fragments 

Some restriction enzymes cleave DNA poorly when 

their recognition sites are located close to DNA 

termini. Table 1.10 lists the activities of Thermo 

Scientific restriction enzymes when their target 

sites are located close to the end of a PCR product. 

Experiments were performed as follows: 

PCR primers were designed with 1-5 extra 

Table 1.10. Cleavage efficiency close to the termini of PCR fragments. 

Enzyme 

bp from the recognition site to fragment end 

1 2 3 4 5 

AanI 0 0-20 20-50 50-100 

AarI 20-50 50-100 

AatII 0 0-20 20-50 50-100 

Acc65I 0-20 50-100 

AdeI 50-100 

AjiI 50-100 

AluI 0-20 20-50 50-100 

Alw21I 50-100 

Alw26I 50-100 

Alw44I 0 20-50 50-100 

ApaI 50-100 

BamHI 50-100 

BauI 0-20 20-50 50-100 

BcnI 20-50 50-100 

BclI 0 50-100 

BcuI 50-100 

BfmI 50-100 

BfuI 50-100 

BglI 20-50 50-100 

BglII 0 50-100 

Bme1390I 20-50 50-100 

BoxI 0 50-100 

BpiI 50-100 

Bpu10I 20-50 50-100 

Bpu1102I 50-100 

BseDI 0 50-100 

BseGI 50-100 

BseJI 0 50-100 

BseLI 0 50-100 

BseMI 0-20 50-100 

BseMII 50-100 

BseNI 0 50-100 

BseSI 50-100 

BseXI 20-50 50-100 

Bsh1236I 50-100 

Bsh1285I 0-20 50-100 

BshNI 50-100 

BshTI 20-50 50-100 

Bsp68I 0 50-100 

Bsp119I 50-100 

Bsp120I 20-50 50-100 

Bsp143I 50-100 

Bsp1407I 20-50 50-100 

BspLI 50-100 

BspOI 0 20-50 50-100 

BspPI 0 50-100 

BspTI 0 0-20 50-100 

Bst1107I 0-20 50-100 

BstXI 0 50-100 

Bsu15I 50-100 

BsuRI 0-20 20-50 50-100 

BveI 0-20 50-100 

CaiI 0 0-20 50-100 

Cfr9I 20-50 50-100 

Cfr10I 20-50 50-100 

Cfr13I 50-100 

Cfr42I 50-100 

CpoI 50-100 

CseI 50-100 

Csp6I 50-100 

DpnI 50-100 

DraI 0 0-20 50-100 

Eam1104I 0 50-100 

Eam1105I 0 50-100 

nucleotides at their 5’-end adjacent to the 

recognition site for the restriction enzyme. 

The 5’-end was labeled with [ 32 P] by T4 

 

labeled primers were used in the PCR reaction. 

PCR products were purified with the Silica Bead 

 

Enzyme 


1 2 3 4 5 

Ecl136II 50-100 

Eco24I 50-100 

Eco31I 20-50 50-100 

Eco32I 20-50 50-100 

Eco47I 50-100 

Eco47III 0 0-20 50-100 

Eco52I 0-20 50-100 

Eco57I 50-100 

Eco72I 0-20 50-100 

Eco81I 50-100 

Eco88I 50-100 

Eco91I 20-50 50-100 

Eco105I 20-50 50-100 

Eco130I 0 50-100 

Eco147I 0 50-100 

EcoO109I 50-100 

EcoRI 50-100 

EcoRII 0 0-20 20-50 50-100 

EheI 20-50 50-100 

Esp3I 50-100 

FaqI 0-20 50-100 

FspAI 0-20 20-50 50-100 

FspBI 20-50 50-100 

GsuI 50-100 

HhaI 50-100 

Hin1I 50-100 

Hin1II 0 0-20 50-100 

Hin6I 0 0-20 50-100 

HincII 50-100 

HindIII 0 0-20 50-100 

HinfI 50-100 

HpaII 0-20 50-100 

HphI 0 50-100 

Hpy8I 0-20 50-100 

HpyF3I 20-50 50-100 

HpyF10VI 50-100 

KpnI 50-100 

Kpn2I 0 50-100 

KspAI 20-50 50-100 

LguI 50-100 

Lsp1109I 0-20 50-100 

LweI 20-50 50-100 

MauBI 0 0-20 20-50 50-100 

MbiI 0 0-20 50-100 

MboI 50-100 

MboII 50-100 

MlsI 0-20 50-100 

MluI 20-50 50-100 

MnlI 0 50-100 

Mph1103I 20-50 50-100 

MreI 0 20-50 50-100 

MspI 0-20 50-100 

MssI 20-50 50-100 

MunI 20-50 50-100 

MvaI 0 20-50 50-100 

Mva1269I 0 50-100 

NcoI 0 50-100 

NdeI* 0-20 20-50 50-100 

NheI 0 20-50 50-100 

NmuCI 20-50 50-100 

NotI 20-50 50-100 

NsbI 0 0-20 50-100 

OliI 0-20 20-50 50-100 

PacI 20-50 50-100 

 

were incubated with 10 units of restriction 

enzyme in the optimal buffer for 1 hour at the 

recommended temperature. Reaction products 

were separated on 10% PAGE and the cleavage 

 

Image Analysis software. 

Enzyme 


1 2 3 4 5 

PaeI 0 0-20 20-50 50-100 

PagI 20-50 50-100 

PasI 50-100 

PauI 0 50-100 

PdiI 0-20 50-100 

PdmI 50-100 

PfeI 50-100 

Pfl23II 0-20 20-50 50-100 

PfoI 0 20-50 50-100 

Ppu21I 50-100 

PscI 0 50-100 

Psp5II 0 50-100 

Psp1406I 0-20 50-100 

PstI 0-20 50-100 

PsuI 0-20 50-100 

PsyI 0 50-100 

PvuI 20-50 50-100 

PvuII 50-100 

RsaI 50-100 

RseI 0 0-20 20-50 50-100 

SacI 50-100 

SalI 20-50 50-100 

SatI 0 50-100 

ScaI 0-20 50-100 

SchI 50-100 

SdaI 0-20 50-100 

SduI 50-100 

SfaAI 0 0-20 20-50 50-100 

SfiI 50-100 

SgrDI 0-20 20-50 50-100 

SgsI 50-100 

SmaI 50-100 

SmiI 0-20 50-100 

SmoI 0 50-100 

SsiI 50-100 

SspI 0-20 50-100 

SspDI 20-50 50-100 

TaaI 20-50 50-100 

TaiI 50-100 

TaqI 0 20-50 50-100 

TasI 0 20-50 50-100 

TatI 0-20 20-50 50-100 

TauI 20-50 50-100 

Tru1I 0 0-20 50-100 

TscAI 0 50-100 

Van91I 0 50-100 

VspI 50-100 

XagI 20-50 50-100 

XapI 20-50 50-100 

XbaI 20-50 50-100 

XceI 0 50-100 

XhoI 0-20 50-100 

XmaJI 50-100 

XmiI 50-100 

I-SceI 50-100 

* – incubation was performed for 16 hours. 

Cleavage efficiency 

0% 20-50% 

0-20% 50-100% 


170 

Cleavage of Restriction Targets Located in Close Vicinity within pUC19 Multiple Cloning Site 

Close vicinity of restriction targets within multiple 

 

effect on the efficiency of double digestions 

within the MSC. For efficient double digestion 

within the MSC, the first reaction should be 

performed with a restriction enzyme that cleaves 

DNA inefficiently when enzyme’s target is 

located close to the end of DNA. The second 

pUC19 multiple cloning site 


digestion should be performed with a restriction 

enzyme which tolerates a close proximity to the 

DNA end. 

The data presented below were generated using 

pUC19 plasmid DNA. The plasmid was cleaved 

 

and end-labeled with [ 32 P] by T4 Polynucleotide 

 

Table 1.11. Cleavage of restriction targets located in close vicinity within pUC19 multiple cloning site. 

Enzyme 

pair 

Acc65I 

BamHI 

Acc65I 

Ecl136II 

Acc65I 

Eco24I 

Acc65I 

EcoRI 

Acc65I 

SacI 

Acc65I 

XapI 

BamHI 

Cfr9I 

BamHI 

Eco88I 

BamHI 

HincII 

BamHI 

KpnI 

BamHI 

SalI 

BamHI 

XbaI 

Cfr9I 

Ecl136I 

Cfr9I 

Eco24I 

Cfr9I 

SacI 

Cfr9I 

XbaI 

Ecl136II 

Eco88I 

Ecl136II 

EcoRI 

Ecl136II 

XapI 

Eco24I 

Eco88I 

Eco24I 

EcoRI 

Eco24I 

KpnI 

Eco24I 

XapI 

Eco88I 

SacI 

Eco88I 

XbaI 

First cut 

Second 

cut 

Efficiency 

of the 2nd 

cut, % 

bp from 

1st cut* 

Acc65I BamHI 100 4 

BamHI Acc65I 100 4 

Acc65I Ecl136II 100 1 

Ecl136II Acc65I 50-100 3 

Acc65I Eco24I 100 1 

Eco24I Acc65I 0 1 

Acc65I EcoRI 100 7 

EcoRI Acc65I 100 7 

Acc65I SacI 50-100 1 

SacI Acc65I 0-20 1 

Acc65I XapI 50-100 7 

XapI Acc65I 50-100 7 

BamHI Cfr9I 50-100 0 

Cfr9I BamHI 50-100 0 

BamHI Eco88I 100 0 

Eco88I BamHI 50-100 0 

BamHI HincII 50-100 7 

HincII BamHI 100 9 

BamHI KpnI 100 4 

KpnI BamHI 100 4 

BamHI SalI 50-100 7 

SalI BamHI 100 7 

BamHI XbaI 20-50 1 

XbaI BamHI 100 1 

Cfr9I Ecl136II 50-100 5 

Ecl136II Cfr9I 100 7 

Cfr9I Eco24I 50-100 5 

Eco24I Cfr9I 100 5 

Cfr9I SacI 50-100 5 

SacI Cfr9I 50-100 5 

Cfr9I XbaI 50-100 6 

XbaI Cfr9I 50-100 6 

Ecl136II Eco88I 100 7 

Eco88I Ecl136II 50-100 5 

Ecl136II EcoRI 100 3 

EcoRI Ecl136II 100 1 

Ecl136II XapI 50-100 3 

XapI Ecl136II 50-100 1 

Eco24I Eco88I 100 5 

Eco88I Eco24I 50-100 5 

Eco24I EcoRI 100 1 

EcoRI Eco24I 100 1 

Eco24I KpnI 50-100 1 

KpnI Eco24I 0 1 

Eco24I XapI 50-100 1 

XapI Eco24I 20-50 1 

Eco88I SacI 100 5 

SacI Eco88I 50-100 5 

Eco88I XbaI 100 6 

XbaI Eco88I 100 6 

Enzyme 

pair 

EcoRI 

Cfr9I 

EcoRI 

Eco88I 

EcoRI 

KpnI 

EcoRI 

SacI 

HincII 

PaeI 

HincII 

PstI 

HincII 

SdaI 

HincII 

XbaI 

HindIII 

PaeI 

HindIII 

PstI 

HindIII 

SdaI 

KpnI 

Ecl136II 

KpnI 

SacI 

KpnI 

XapI 

PaeI 

PstI 

PaeI 

SalI 

PaeI 

SdaI 

PstI 

BamHI 

PstI 

SalI 

PstI 

XbaI 

SacI 

SmaI 

SacI 

XapI 

SalI 

SdaI 

SalI 

XbaI 

SdaI 

XbaI 

First cut 

Second 

cut 

Efficiency 

of the 2nd 

cut, % 

bp from 

1st cut* 

EcoRI Cfr9I 50-100 11 

Cfr9I EcoRI 100 11 

EcoRI Eco88I 50-100 11 

Eco88I EcoRI 100 11 

EcoRI KpnI 100 7 

KpnI EcoRI 100 7 

EcoRI SacI 50-100 1 

SacI EcoRI 20-50 1 

HincII PaeI 100 9 

PaeI HincII 100 7 

HincII PstI 100 3 

PstI HincII 20-50 1 

HincII SdaI 0-20 2 

SdaI HincII 20-50 1 

HincII XbaI 50-100 3 

XbaI HincII 50-100 1 

HindIII PaeI 50-100 1 

PaeI HindIII 0 1 

HindIII PstI 100 7 

PstI HindIII 100 7 

HindIII SdaI 50-100 6 

SdaI HindIII 50-100 7 

KpnI Ecl136II 50-100 1 

Ecl136II KpnI 100 3 

KpnI SacI 50-100 1 

SacI KpnI 100 1 

KpnI XapI 100 7 

XapI KpnI 50-100 7 

PaeI PstI 0 1 

PstI PaeI 20-50 1 

PaeI SalI 50-100 7 

SalI PaeI 100 7 

PaeI SdaI 0 0 

SdaI PaeI 20-50 1 

PstI BamHI 50-100 13 

BamHI PstI 50-100 13 

PstI SalI 0 1 

SalI PstI 50-100 1 

PstI XbaI 100 7 

XbaI PstI 50-100 7 

SacI SmaI 100 5 

SmaI SacI 50-100 7 

SacI XapI 0-20 1 

XapI SacI 50-100 1 

SalI SdaI 0 0 

SdaI SalI 20-50 1 

SalI XbaI 50-100 1 

XbaI SalI 0-20 1 

SdaI XbaI 100 7 

XbaI SdaI 50-100 6 

for one hour with a second restriction enzyme 

 

separated by PAGE, and the amount of the 

label present on the DNA was determined by 

autoradiography. A decrease in radioactivity 

reflects cleavage efficiency of the second 

restriction enzyme. 

Eco24I Cfr9I HincII PstII 

XapI Ecl136II Acc65I Eco88I SalI BveI PaeI 

396 EcoRI SacI KpnI SmaI BamHI XbaI XmiI SdaI HindIII 452 

5’ - C CAG TGA ATT CGA GCT CGG TAC CCG GGG ATC CTC TAG AGT CGA CCT GCA GGC ATG CAA GCT TGG C - 3’ 

3’ - G GTC ACT TAA GCT CGA GCC ATG GGC CCC TAG CAG ATC TCA GCT GGA CGT CCG TAC GTT CGA ACC G - 5’ 

Enzyme 

pair 

SmaI 

Acc65I 

SmaI 

BamHI 

SmaI 

Ecl136II 

SmaI 

Eco24I 

SmaI 

KpnI 

SmaI 

XbaI 

First cut 

Second 

cut 

Efficiency 

of the 2nd 

cut, % 

* – only double-stranded portion of DNA is 

included, not the overhangs. 

– simultaneous double digestion may be 

performed with these enzyme pairs. 

– this enzyme should be used for the first cut 

– this enzyme should be used for the second cut 

– not recommended 

For reaction conditions see 

www.thermoscientific.com/doubledigest 

bp from 

1st cut* 

SmaI Acc65I 0-20 1 

Acc65I SmaI 0 -1 

SmaI BamHI 50-100 2 

BamHI SmaI 0-20 0 

SmaI Ecl136II 50-100 7 

Ecl136II SmaI 100 7 

SmaI Eco24I 50-100 7 

Eco24I SmaI 100 5 

SmaI KpnI 0 1 

KpnI SmaI 0 -1 

SmaI XbaI 50-100 8 

XbaI SmaI 100 6

Common Properties 

Classification of Restriction 

Enzymes 

Restriction enzymes recognize specific nucleotide 

sequences and cleave DNA molecules either 

within or outside their recognition site. Digestion 

results in the generation of DNA with “sticky” 

 

The phenomenon of host specificity was first 

observed by Luria and Human in the early 

 

 

proposed that host specificity is based on a 

two-enzyme system: a restriction enzyme, 

which recognizes specific DNA sequences and 

cleaves foreign DNA upon its entrance into 

the bacterial cell, and a modification enzyme 

 

DNA from degradation by its own restriction 

enzyme. Both restriction and modification 

enzymes recognize the same nucleotide 

sequence and together they form a restriction- 

 

Restriction-modification systems are 

widespread among bacteria and have also 

been isolated from phage, Archaea and 

eukaryotic algae viruses. R-M systems have 

 

depending on the complexity of their structure, 

cofactor requirements, mode of action and 

 

and the most frequently used are type II 

restriction endonucleases. These enzymes 

recognize specific 4-8 bp DNA sequences 

and cleave a DNA sequence either within the 

recognition site, or at a specified position up to 

20 base pairs outside the site. 

Often there is more than one enzyme that 

recognizes a particular nucleotide sequence. 

According to restriction enzyme nomenclature, 

an enzyme with a unique specificity which has 

been discovered first is called the prototype. 

Subsequently discovered enzymes with the 

same specificity are termed isoschizomers. 

An isoschizomer may differ from the prototype 

enzyme in site preferences, reaction conditions, 

as well as in sensitivity to methylation and 

exhibition of star activity. Restriction enzymes 

that recognize the same nucleotide sequence, 

but cleave DNA at different positions are called 

neoschizomers. 

Site Preferences 

by Restriction Enzymes 

In 1975, Thomas and Davis discovered that 

EcoRI cleaves its five recognition sites on DNA 

at rates that differ by an order of magnitude 

 

for other restriction enzymes. Factors such as 

flanking sequences and the number of cleavage 

 

 

 

never completely digest certain unmethylated 

target DNAs, even when using an excess of 

 

these enzymes are members of the expanding 

group of type II restriction enzymes which 

require simultaneous interaction with two copies 

 

These enzymes cleave DNA molecules with one 

recognition site very slowly. In the case of type 

 

sequences serves as an allosteric effector 

for effective cleavage of the other recognition 

 

 

 

 

 

Type IIS enzymes, such as FokI, BpmI, BsgI 

and MboII, also interact with two copies of their 

recognition sequence before cleaving DNA by 

 

Cleavage of resistant sites can be significantly 

enhanced by the addition of cleavable DNA, 

an oligodeoxyribonucleotide containing the 

 

 

Different restriction enzymes recognizing the 

 

or 

cleave the same resistant recognition site 

 

Eam1104I and PdiI and their prototypes 

 

some isoschizomers or neoschizomers cleave 

“resistant” sites at the same rate as normal 

sites. For example, EheI cleaves target DNA 

more efficiently than its prototype NarI. Thus, 

one recognition site of NarI onDNA and two 

sites on pBR322 are not cleaved to completion, 

even after incubation with 50 units of the 

enzyme for 16 hours. Unlike NarI, Thermo 

Scientific neoschizomer EheI cleaves DNA 

and pBR322 DNA completely under standard 

conditions. 

References 

1. Luria, S.E., Human, M.L., A nonhereditary, host-induced 

 

557-569, 1952. 

2. Arber, W., Dussoix, D., Host specificity of DNA produced 

by Escherichia Coli: I. Host controlled modification of 

 

3. 

enzymes, DNA methyltransferases, homing endonucleases 

and their genes, Nucleic Acids Res., 31, 

1805-1812, 2003. 

4. Thomas M., Davis R.W., Studies on the cleavage of 

bacteriophage lambda DNA with EcoRI restriction 

 

5. 

the relaxed DNA sequence specificity of “promiscuous” 

 

307-324, 2005. 

6. Kruger, D.H., et al., EcoRII can be activated to cleave 

refractory DNA recognition sites, Nucleic Acids Res., 16, 

3997-4008, 1988. 

7. Oller, A. R., et al., Ability of DNA and spermidine to affect 

the activity of restriction endonucleases from several 

bacterial species, Biochemistry, 30, 2543-2549, 1991. 

8. 

endonuclease from Kluyvera species 632 with the 

asymmetric hexanucleotide recognition sequence: 

5’-CTCTTCN-3’ 3’-GAGAAGNNNN-5’, Gene, 66, 

31-43, 1988. 

9. Reuter, M., et al., Use of specific oligonucleotide duplexes 

to stimulate cleavage of refractory DNA sites by restriction 

endonucleases, Anal. Biochem., 209, 232-237, 1993. 

10. Halford, S.E., Hopping, jumping and looping by restriction 

endonucleases, Biochem. Soc. Trans, 29, 363-373, 2001. 

11. Gabbara, S., Bhagwat, A.S., Interaction of EcoRII 

endonuclease with DNA substrates containing single 

 

12. Yang, C.C., Topal, M.D., Nonidentical DNA-binding sites 

of endonuclease NaeI recognize different families of 

sequences flanking the recognition site, Biochemistry, 31, 

9657-9664, 1992. 

13. 

bridge between DNA endonuclease and topoisomerase, 

 

14. Wentzell, L.M., et al., The SfiI restriction endonuclease 

makes a four-strand DNA break at two copies of its 

 

15. Siksnys, V., et al., The Cfr10I restriction enzyme is 

 

1105-1118, 1999. 

16. Deibert, M., et al., Structure of the tetrameric restriction 

endonuclease NgoMIV in complex with cleaved DNA, Nat. 

Struct. Biol., 7, 792-799, 2000. 

17. Gormley, N.A., et al., The type IIs restriction endonuclease 

BspMI is a tetramer that acts concertedly at two copies of 

 

4034-4041, 2002. 

18. 

and lexcise two recognition sites from DNA, 

 

19. 

interact with two recognition sites before cleaving DNA, 

 

20. Conrad, M., Topal, M.D., DNA and spermidine provide a switch 

mechanism to regulate the activity of restriction enzyme NaeI, 

Proc. Natl.Acad Sci. USA, 86, 9707-9711, 1989. 

21. 

Arthrobacter aurescens SS2-322, which recognizes the 

4/8-3’, 

Nucleic Acids Res., 30, e123, 2002. 


172 

Star Activity 

Restriction enzymes recognize specific 

nucleotide sequences within DNA molecules. 

However, their recognition site specificity 

can be reduced in vitro 

conditions, enzymes are able to recognize and 

cleave nucleotide sequences which differ from 

the canonical site. At low ionic strength, for 

 

 

sequences: NGATCC, GPuATCC and GGNTCC 

 

 

applications, star activity is not desirable. 

 

suggested that this phenomenon is the result of: 

 

high enzyme concentration in the reaction 

 

high glycerol concentration in the 

reaction mixture, 

presence of organic solvents, such as 

 

 

reaction mixture, 

low ionic strength of the reaction buffer, 

suboptimal pH values of the 

reaction buffer, 

substitution of Mg 2+ for other divalent 

cations, such as Mn 2+ or Co 2+ . 


In some cases, the termini generated by DNA 

cleavage with a restriction enzyme at the 

canonical site have been shown to stimulate 

 

Both star activity and incomplete DNA digestion 

result in atypical electrophoresis patterns that 

can be distinguished by careful examination of 

see 

Any tendency of a restriction enzyme to exhibit 

star activity is indicated both in the product 

description and in the Certificate of Analysis. 

How to distinguish between star activity and incomplete digestion 

Star activity results in additional DNA 

bands below the expected bands and 

no additional bands above the largest 

expected fragment. These additional bands 

become more intense, while the expected 

bands become less intense, when either 

the incubation time or the amount of 

enzyme is increased. 

Incomplete DNA digestion results in 

additional bands above the expected 

DNA bands on the gel. Additional bands 

disappear when the incubation time 

or amount of enzyme is increased. No 

additional bands below the smallest 

expected fragment are observed. 

Note 

Certain restriction enzymes remain associated 

with the substrate DNA after cleavage and 

cause a change in the mobility of the digestion 

products during electrophoresis. The resulting 

atypical pattern is not related to star activity. 

In these cases, use a loading dye with SDS 

 

heat the sample for 10 min at 65°C and chill on 

see 

effect is characteristic of the following 

FastDigest enzymes: 

FastDigest 

FastDigest FokI, 

FastDigest MboII, FastDigest TauI, FastDigest 

 

Conventional restriction enzymes: 

 

 

 

 

References 

1. Nasri, M., Thomas, D., Relaxation of recognition sequence 

of specific endonuclease HindIII, Nucleic Acids Res., 14, 

811-821, 1986. 

 

endonuclease BamHI: Relaxation of sequence recognition, 

Proc. Natl. Acad. Sci. USA, 79, 2432-2436, 1982. 

3. Kolesnikov, V.A., et al., Relaxed specificity of endonuclease 

BamHI as determined by identification of recognition sites 

in SV40 and pBR322 DNAs, FEBS Letters, 132, 101-103, 

1981. 

4. Polisky, B., et al., Specificity of substrate recognition by 

the EcoRI restriction endonuclease, Proc. Natl. Acad. Sci. 

USA, 72, 3310-3314, 1975. 

5. Woodbury, C.P., et al., DNA site recognition and reduced 

 

11534-11546, 1980. 

 

 

7. Malyguine, E., et al., Alteration of the specificity of 

restriction endonucleases in the presence of organic 

solvents, Gene, 8, 163-177, 1980. 

8. Hsu, M., Berg, P., Altering the specificity of restriction 

endonuclease: effect of replacing Mg 2+ with Mn 2+ , 

Biochemistry, 17, 131-138, 1978. 

9. Mayer, H., Optimization of the EcoRI* activity of EcoRI 

endonuclease, FEBS Letters, 90, 341-344, 1978. 

10. Nasri, M., Thomas, D., Alteration of the specificity of PvuII 

restriction endonuclease, Nucleic Acids Res., 15, 7677- 

7687, 1987. 

 

relax the specificity of SgrAI restriction endonuclease, 

Proc. Natl. Acad. Sci. USA, 99, 1164-1169, 2002. 

Complete digestion 

Incomplete cleavage Star activity 

1 2 3 4 5 6 

Enzyme complete digestion and star activity. 

1 – Lambda DNA. 

2 – Lambda DNA incubated 1 hour with 0.15 u of 

 

3 – Lambda DNA incubated 1 hour with 0.4 u of 

 

4 – Lambda DNA incubated 1 hour with 1 u of 

 

5 – Lambda DNA incubated 16 hours with 40 u of 

 

6 – Lambda DNA incubated 16 hours with 70 u of

Digestion of Methylated DNA 

DNA methylation is the process of transferring 

a methyl group from a donor molecule to 

either a cytosine or an adenine by DNA 

methyltransferases. Such methylation is the 

most common and abundant DNA modification 

process in living organisms. Three types of 

methylated bases are predominantly 

found in DNA: 

 

 

 

Other modified bases, such as 

 

 

described. The organism-specific pattern of 

methylation depends on the methyltransferases’ 

specificity. 

In prokaryotes, DNA cleavage by a cognate 

restriction enzyme is prevented by the 

methylation of DNA by a sequence-specific 

methyltransferase which is an integral 

component of every restriction-modification 

 

The majority of E.coli strains used for propagation 

of plasmid DNA contain two site-specific DNA 

 

methylase encoded by the dam gene methylates 

the N6-position of an adenine residue within the 

 

by the dcm gene methylates the C5-position of 

the internal cytosine residue within the CCWGG 

 

In addition to Dam and Dcm methylases, 

laboratory strains of E.coli K12 and B may 

contain EcoKI or EcoBI enzymes, respectively, 

encoded by a type I R-M system. These 

methyltransferases modify adenine residues 

within their respective recognition sequences: 

6 8TGCT 

 

DNA from higher eukaryotic organisms 

possesses modified 5-methylcytosine residues 

 

tissue-specific methylation patterns are heritable. 

They participate in regulation of gene expression 

and cellular differentiation. 

In cases where a restriction enzyme target 

site overlaps a methylation site, the following 

digestion results are possible: 

no effect 

partial inhibition 

complete block 

The ability to cleave methylated DNA is an 

intrinsic and unpredictable property of each 

restriction enzyme. Therefore, isoschizomers 

and neoschizomers which recognize the same 

DNA sequences can differ in their sensitivity 

 

 

 

while the isoschizomer Bsp143I is insensitive to 

Dam methylation. EcoRII does not cleave DNA 

at the CCWGG site if it is methylated by Dcm, 

while its neoisoschizomer MvaI will cleave this 

 

DNA digestion, it is necessary to take into 

account both the type of DNA methylation and 

the sensitivity of the restriction enzyme to that 

type of methylation. 

All our restriction enzymes have been examined 

for their sensitivity to Dam, Dcm, CpG, EcoKI and 

EcoBI methylation of substrate DNA. Detailed 

methylation sensitivity information for our 

restriction enzymes is listed in the tables on 

pp.174-179, as well as in the product 

descriptions and Certificates of Analysis. 

Table 1.12. Thermo Scientific isoschizomers and neoschizomers with differing sensitivities to target methylation. 

Conventional 


couple 

Recognition and 

cleavage sites 

Sensitivity to methylation 

Acc65I GGTACC Overlapping Dcm or CpG methylation may influence DNA cleavage 

KpnI GGTACC Not influenced by Dcm or CpG methylation 

ApaI GGGCCC Overlapping Dcm or CpG methylation may influence DNA cleavage 

Bsp120I GGGCCC Blocked by overlapping Dcm or CpG methylation 

Bsp143I GATC Not influenced by Dam, blocked by CpG methylation 

MboI GATC Blocked by Dam methylated DNA 

DpnI GATC Cleaves only Dam methylated DNA 

BspOI GCTAGC Not influenced by CpG methylation 

NheI GCTAGC Overlapping CpG methylation may influence DNA cleavage 

Cfr9I CCCGGG CpG methylation may influence DNA cleavage 

SmaI CCCGGG Blocked by CpG methylation 

Csp6I GTAC Not influenced by CpG methylation 

RsaI GTAC Overlapping CpG methylation may influence DNA cleavage 

Ecl136II GAGCTC Overlapping CpG methylation may influence DNA cleavage 

SacI GAGCTC Not influenced by CpG methylation 

EcoRII CCWGG Blocked by Dcm methylation 

MvaI CCWGG Not influenced by Dcm methylation 

HpaII CCGG Blocked by CpG methylation 

MspI CCGG Not influenced by CpG methylation 

References 

1. Luria, S.E., Human, M.L., A nonhereditary, host-induced 

 

2. Arber, W., Dussoix, D., Host specificity of DNA produced 

by Escherichia Coli: I. Host controlled modification of 

 

3. 

DNA methyltransferases, homing endonucleases and their 

genes, Nucleic Acids Res., 31, 1805-1812, 2003. 

4. McClelland, M., The effect of sequence specific DNA 

methylation on restriction endonuclease cleavage, Nucleic 

Acids Res., 9, 5859-5866, 1981. 

5. McClelland, M., et al., Effect of site-specific modification 

on restriction endonucleases and DNA modification 

methyltransferases, Nucleic Acids Res., 22, 

3640-3659, 1994. 

6. Marinus, M.G., Morris, N.R., Isolation of deoxyribonucleic 

acid methylase mutants of Escherichia coli K-12, 

 

7. May, M.S., Hattman, S., Analysis of bacteriophage 

deoxyribonucleic acid sequences methylated by host- and 

 

768-770, 1975. 

8. Hattman, S., et al., Sequence specificity of the P1 

 

Escherichia coli dam gene, 

 

9. Geier, G.E., Modrich, P., Recognition sequence of the dam 

methylase of Escherichia coli K12 and mode of cleavage 

 

1408-1413, 1979. 

10. Buryanov, Ya.I., et al., Site specificity and chromatographic 

properties of E.coli K12 and EcoRII DNA-cytosine 

methylases, FEBS Letters, 88, 251-254, 1978. 

11. Waalwijk, C., Flavell, R.A., MspI, an isochizomer of HpaII 

which cleaves both unmethylated and methylated HpaII 

sites, Nucleic Acids Res., 5, 3231-3236, 1978. 

12. Bird, A.P., et al., Methylated and unmethylated DNA 

compartments in the sea urchin genome, Cell, 17, 

889-902, 1979. 

13. McClelland, M., The frequency and distribution of 

methylatable DNA sequences in leguminous plant protein 

 

14. Dreiseikelmann, B., et al., The effect of differential 

methylation by Escherichia coli of plasmid DNA and 

phage T7 and lambda DNA on the cleavage by restriction 

endonuclease MboI from Moraxella bovis, Biochim. 

Biophys. Acta, 562, 418-428, 1979. 

15. Butkus, V. et al., Investigation of restriction-modification 

enzymes from M.varians RFL19 with a new type of 

specificity toward modification of substrate, Nucleic Acids 

Res., 13, 5727-5746, 1985. 







S = C or G; 


174 

Effect of Dam Methylation on DNA Cleavage 

To cleave with a restriction enzyme which 

is sensitive to the Dam methylation, DNA 

should be purified from dam – E.coli strains; 

e.g., GM2163. Control digestions should be 

performed with dam – , dcm – 

#SD0021. 

Table 1.13. Completely overlapping 

Dam methyltransferase and restriction enzyme 

recognition sites. 

Conventional 

FastDigest 

Sequence* Effect 

BamHI 

BamHI 

GGm6ATCC 

BclI 

BclI 

TGm6ATCA 

BglII 

BglII 

AGm6ATCT 

Bsp143I 

Sau3AI 

Gm6ATC 

BspPI 

GGm6ATC 

DpnI** 

DpnI 

MboI 

MboI 

PsuI 

PsuI 

PvuI 

PvuI 

SfaAI 

AsiSI 

Gm6ATC 

Gm6ATC 


RGm6ATCY 

CGm6ATCG 

GCGm6ATCGC 

Table 1.14. Partially overlapping 

Dam methyltransferase and restriction enzyme 


Conventional 

Sequence* 

FastDigest 

Effect 

AloI 

5’...GAAC(N) 4Gm6A TCC...3’ 

3’...CTTG(N) 4C Tm6AGG...5’ ? 

BseJI 

BsaBI 

Bsh1285I 

BsiEI 

Bsp68I 

NruI 

Bsu15I 

ClaI 

HphI 

Kpn2I 

Kpn2I 

MboII 

MboII 

PagI 

BspHI 

PfoI 

PfoI 

SmoI 

TaqI 

TaqI 

XbaI 

XbaI 

Note 

** – DpnI Dam 

methylated DNA. 

* – recognition sequence is indicated in bold. 

m6A = N6-methyladenine. 

– overlapping methylase sequences. 

– cleavage not blocked. 

– cleavage blocked. 

5’...Gm6A TC(N) 3ATC...3’ 

3’...C Tm6AG(N) 3TAG...5’ 

5’...CGm6A TCG...3’ 

3’...GC Tm6AGC...5’ 

5’...Gm6A TCGCGm6A TC...3’ 

3’...C Tm6AGCGC Tm6AG...5’ 

5’...ATCGm6A TC...3’ 

3’...TAGC Tm6AG...5’ 

5’...GGTGm6A TC...3’ 

3’...CCAC Tm6AG...5’ 

5’...TCCGGm6A TC...3’ 

3’...AGGCC Tm6AG...5’ 

5’...Gm6A TCCGGm6A TC...3’ 

3’...C Tm6AGGCC Tm6AG...5’ 

5’...GAAGm6A TC...3’ 

3’...CTTC Tm6AG...5’ 

5’...TCATGm6A TC...3’ 

3’...AGTAC Tm6AG...5’ 

5’...Gm6A TCATGm6A TC...3’ 

3’...C Tm6AGTAC Tm6AG...5’ 

5’...TCCNGGm6A TC...3’ 

3’...AGGNCC Tm6AG...5’ 

5’...CTYRAGm6A TC...3’ 

3’...GARYTC Tm6AG...5’ 

5’...TCGm6A TC...3’ 

3’...AGC Tm6AG...5’ 

5’...TCTAGm6A TC...3’ 

3’...AGATC Tm6AG...5’ 

– cleavage rate is slowed significantly by 

methylation. 

? – the sensitivity to methylation has not been 

determined. 

? 

? 







S = C or G;

Effect of Dcm Methylation on DNA Cleavage 

To cleave with a restriction enzyme which is 

sensitive to Dcm methylation, DNA should be 

purified from dcm – E.coli strains; e.g., GM2163. 

Control digestions should be performed with 


Table 1.15. Completely overlapping 

Dcm methyltransferase and restriction 

enzyme recognition sites. 

Conventional 

FastDigest Sequence* Effect 

EcoRII Cm5CWGG 

MvaI 

MvaI 







S = C or G; 

Note 


** – SgeI cleaves only methylated DNA. 


m5C = 5-methylcytosine. 



Cm5CWGG 

PasI CCm5CWGGG 

SexAI ACm5CWGGT 

SgeI** Cm5CWGG 

– cleavage rate is slowed significantly by 

methylation. 

Table 1.16. Partially overlapping 

Dcm methyltransferase and restriction enzyme 


Conventional 

FastDigest 

Acc65I 

Acc65I 

AloI 

ApaI 

ApaI 

BamHI 

BamHI 

BfuI 

BglI 

BglI 

Bme1390I 

ScrFI 

BseDI 

BsaJI 

BseGI 

BseGI 

BseLI 

BslI 

BseSI 

Bme1580I 

BshNI 

BanI 

Bsp120I 

Bsp120I 

BspLI 

NlaIV 

BspPI 

BstXI 

BstXI 

BsuRI 

HaeIII 

CaiI 

AlwNI 

Cfr13I 

Sau96I 


5’...GGTACm5CW GG...3’ 

3’...CCATG GWm5CC...5’ 

5’...Cm5CW GGTACm5CW GG...3’ 

3’...G GWm5CCATG GWm5CC...5’ 

5’...GAAC(N) 6TCm5CW GG...3’ 

3’...CTTG(N) 6AG GWm5CC...5’ 

5’...GGGCCm5CW GG...3’ 

3’...CCCGG GWm5CC...5’ 

5’...Cm5CW GGATCm5CW GG...3’ 

3’...G GWm5CCTAG GWm5CC...5’ 

5’...GTATCm5CW GG...3’ 

3’...CATAG GWm5CC...5’ 

5’...GCm5CW GGNNGGC...3’ 

3’...CG GWm5CCNNCCG...5’ 

5’...Cm5CW GG...3’ 

3’...G GWm5CC...5’ 

5’...Cm5CW GGG...3’ 

3’...G GWm5CCC...5’ 

5’...Cm5CW GGATG...3’ 

3’...G GWm5CCTAC...5’ 

5’...Cm5CW GG(N) 4GG...3’ 

3’...G GWm5CC(N) 4CC...5’ 

5’...Cm5CW GGNCm5CW GG...3’ 

3’...G GWm5CCNG GWm5CC...5’ 

5’...GKGCCm5CW GG...3’ 

3’...CMCGG GWm5CC...5’ 

5’...GGYRCm5CW GG...3’ 

3’...CCRYG GWm5CC...5’ 

5’...Cm5CW GGYRCm5CW GG...3’ 

3’...G GWm5CCRYG GWm5CC...5’ 

5’...GGGCCm5CW GG...3’ 

3’...CCCGG GWm5CC...5’ 

5’...GGNNCm5CW GG...3’ 

3’...CCNNG GWm5CC...5’ 

5’...Cm5CW GGNNCm5CW GG...3’ 

3’...G GWm5CCNNG GWm5CC...5’ 

5’...Cm5CW GGATC...3’ 

3’...G GWm5CCTAG...5’ 

5’...Cm5CA GG(N) 4TGG...3’ 

3’...G GTm5CC(N) 4ACC...5’ 

5’...Cm5CA GGNNCm5CT GG...3’ 

3’...G GTm5CCNNG GAm5CC...5’ 

5’...GGCm5CW GG...3’ 

3’...CCG GWm5CC...5’ 

5’...Cm5CW GGCm5CW GG...3’ 

3’...G GWm5CCG GWm5CC...5’ 

5’...CAGNNCm5CT GG...3’ 

3’...GTCNNG GTm5CC...5’ 

5’...GGNCm5CW GG...3’ 

3’...CCNG GWm5CC...5’ 

Conventional 

FastDigest 

Eco24I 

Eco31I 

Eco31I 

Eco47I 

AvaII 

Eco91I 

Eco91I 

Eco147I 

StuI 

EcoO109I 

EcoO109I 

EheI 

EheI 

FaqI 

BsmFI 

FokI 

GsuI 

BpmI 

Hin1I 

BsaHI 

HphI 

KpnI 

KpnI 

MlsI 

MscI 

PfoI 

PfoI 

Psp5II 

PpuMI 

SduI 

Bsp1286I 

SfiI 

SfiI 

SgsI 

AscI 

SspDI 

Van91I 

PflMI 

XagI 

EcoNI 


5’...Cm5CW GGGCCm5CW GG...3’ 

3’...G GWm5CCCGG GWm5CC...5’ 

5’...Cm5CW GGTCTC...3’ 

3’...G GWm5CCAGAG...5’ 

5’...GGWCm5CW GG...3’ 

3’...CCWG GWm5CC...5’ 

5’...Cm5CW GGTNACm5CW GG...3’ 

3’...G GWm5CCANTG GWm5CC...5’ 

5’...AGGCm5CT GG...3’ 

3’...TCCG GAm5CC...5’ 

5’...RGGNCm5CT GG...3’ 

3’...YCCNG GAm5CC...5’ 

5’...Cm5CW GGCGCm5CW GG...3’ 

3’...G GWm5CCGCG GWm5CC...5’ 

5’...Cm5CW GGGAC...3’ 

3’...G GWm5CCCTG...5’ 

5’...Cm5CW GGATG...3’ 

3’...G GWm5CCTAC...5’ 

5’...CTCm5CA GG...3’ 

3’...GAG GTm5CC...5’ 

5’...GRCGCm5CW GG...3’ 

3’...CYGCG GWm5CC...5’ 

5’...Cm5CW GGTGA...3’ 

3’...G GWm5CCACT...5’ 

5’...GGTACm5CW GG...3’ 

3’...CCATG GWm5CC...5’ 

5’...Cm5CW GGTACm5CW GG...3’ 

3’...G GWm5CCATG GWm5CC...5’ 

5’...TGGCm5CA GG...3’ 

3’...ACCG GTm5CC...5’ 

5’...TCm5CA GGA...3’ 

3’...AG GTm5CCT...5’ 

5’...RGGWCm5CT GG...3’ 

3’...YCCWG GAm5CC...5’ 

5’...Cm5CW GGGCCm5CW GG...3’ 

3’...G GWm5CCCGG GWm5CC...5’ 

5’...GGCm5CW GGNNGGCC...3’ 

3’...CCG GWm5CCNNCCGG...5’ 

5’...Cm5CW GGCC(N) 5GGCm5CW GG...3’ 

3’...G GWm5CCGG(N) 5CCG GWm5CC...5’ 

5’...Cm5CW GGCGCGCm5CW GG...3’ 

3’...G GWm5CCGCGCG GWm5CC...5’ 

5’...Cm5CW GGCGCC...3’ 

3’...G GWm5CCGCGG...5’ 

5’...Cm5CA GG(N) 3TGG...3’ 

3’...G GTm5CC(N) 3ACC...5’ 

5’...Cm5CT GG(N) 3AGG...3’ 

3’...G GAm5CC(N) 3TCC...5’ 

5’...Cm5CT GGNCm5CA GG...3’ 

3’...G GAm5CCNG GTm5CC...5’ 


176 

Effect of CpG Methylation on DNA Cleavage 

Methylated DNA substrates were prepared using SssI methyltransferase. 

Table 1.17. CpG is located inside the recognition site. 

Conventional 

FastDigest 

Sequence Effect 

AatII 

AatII 

GACGTC 

AjiI CACGTC 

BauI CACGAG 

BcnI 

NciI 

Bsh1236I 

Bsh1236I 

Bsh1285I 

BsiEI 

BshTI 

AgeI 

Bsp68I 

NruI 

Bsp119I 

Bsp119I 

Bsu15I 

ClaI 

CCSGG 

CGCG 

CGRYCG 

ACCGGT 

TCGCGA 

TTCGAA 

ATCGAT 

Cfr9I CCCGGG 

Cfr10I 

BsrFI 

RCCGGY 

Cfr42I CCGCGG 

CpoI 

RsrII 

CseI 

HgaI 

Eco47III 

AfeI 

Eco52I 

EagI 

Eco72I 

PmlI 

Eco88I 

AvaI 

Eco105I 

SnaBI 

EheI 

EheI 

Esp3I 

BsmBI 

FspAI 

FspAI 

CGGWCCG 

GACGC 

AGCGCT 

CGGCCG 

CACGTG 

CYCGRG 

TACGTA 

GGCGCC 

CGTCTC 

RTGCGCAY 

HaeII RGCGCY 

HhaI 

HhaI 

Hin1I 

BsaHI 

Hin6I 

HinP1I 

GCGC 

GRCGYC 

GCGC 


Conventional 

FastDigest 

Sequence Effect 

HpaII 

HpaII 

CCGG 

Kpn2I 

Kpn2I 

TCCGGA 

MauBI 

MauBI 

CGCGCGCG 

MbiI 

BsrBI 

GAGCGG 

MluI 

MluI 

ACGCGT 

MreI 

MreI 

CGCCGGCG 

MspI 

MspI 

CCGG 

NotI 

NotI 

GCGGCCGC 

NsbI 

FspI 

TGCGCA 

PauI 

BssHII 

GCGCGC 

PdiI 

NaeI 

GCCGGC 

Pfl23II 

BsiWI 

CGTACG 

Ppu21I 

BsaAI 

YACGTR 

Psp1406I 

AclI 

AACGTT 

PvuI 

PvuI 

CGATCG 

SalI 

SalI 

GTCGAC 

SfaAI 

AsiSI 

GCGATCGC 

SgrDI CGTCGACG 

SgsI 

AscI 

SmaI 

SmaI 

SsiI 

AciI 

GGCGCGCC 

CCCGGG 

CCGC 

SspDI GGCGCC 

TaiI 

TaiI 

TaqI 

TaqI 

ACGT 

TCGA 

TauI GCSGC 

XhoI 

XhoI 

Note 

CTCGAG 



– cleavage rate is reduced significantly by 

methylation. 







S = C or G;

Table 1.18. CpG partially overlaps the recognition site. 

Conventional 

FastDigest 

AarI 

DrdI 

Acc65I 

Acc65I 

AdeI 

DraIII 

AjuI 

AjuI 

AlfI 

AloI 

Alw21I 

Alw21I 

Alw26I 

Alw26I 

Alw44I 

ApaLI 

ApaI 

ApaI 

BamHI 

BamHI 

BfuI 

BglI 

BglI 

Bme1390I 

ScrFI 


5’...CACCTGm5C G...3’ 

3’...GTGGAC Gm5C...5’ 

5’...m5C GAC(N) 6GTC...3’ 

3’... Gm5CTG(N) 6CAG...5’ 

5’...m5C GAC(N) 6GTm5C G...3’ 

3’... Gm5CTG(N) 6CA Gm5C...5’ 

5’...GAm5C G(N) 5GTC...3’ 

3’...CT Gm5C(N) 5CAG...5’ 

5’...GAm5C G(N) 4m5C GTC...3’ 

3’...CT Gm5C(N) 4 Gm5CAG...5’ 

5’...GGTACm5C G...3’ 

3’...CCATG Gm5C...5’ 

5’...m5C GGTACm5C G...3’ 

3’... Gm5CCATG Gm5C...5’ 

5’...CAm5C GNNGTG...3’ 

3’...GT Gm5CNNCAC...5’ 

5’...CAm5C GNm5C GTG...3’ 

3’...GT Gm5CN Gm5CAC...5’ 

5’...m5C GAA(N) 7TTGG...3’ 

3’... Gm5CTT(N) 7AACC...5’ 

5’...GCA(N) 6TGm5C G...3’ 

3’...CGT(N) 6AC Gm5C...5’ 

5’...m5C GAAC(N) 6TCC...3’ 

3’... Gm5CTTG(N) 6AGG...5’ 

5’...GAAC(N) 6TCm5C G...3’ 

3’...CTTG(N) 6AG Gm5C...5’ 

5’...GAAm5C G(N) 5TCC...3’ 

3’...CTT Gm5C(N) 5AGG...5’ 

5’...m5C GWGCWm5C G...3’ 

3’... Gm5CWCGW Gm5C...5’ 

5’...GTCTm5C G...3’ 

3’...CAGA Gm5C...5’ 

5’...m5C GTCTC...3’ 

3’... Gm5CAGAG...5’ 

5’...GTGCAm5C G...3’ 

3’...CACGT Gm5C...5’ 

5’...GGGCCm5C G...3’ 

3’...CCCGG Gm5C...5’ 

5’...m5C GGGCCm5C G...3’ 

3’... Gm5CCCGG Gm5C...5’ 

5’...m5C GGATCm5C G...3’ 

3’... Gm5CCTAG Gm5C...5’ 

5’...m5C GTATCC...3’ 

3’... Gm5CATAGG...5’ 

5’...GTATCm5C G...3’ 

3’...CATAG Gm5C...5’ 

5’...GCm5C G(N) 3m5C GGC...3’ 

3’...CG Gm5C(N) 3 Gm5CCG...5’ 

5’...GCC(N) 5GGm5C G...3’ 

3’...CGG(N) 5CC Gm5C...5’ 

5’...m5C GCC(N) 5GGm5C G...3’ 

3’... Gm5CGG(N) 5CC Gm5C...5’ 

5’...Cm5C GGG...3’ 

3’...G Gm5CCC...5’ 

? 

? 

Conventional 

FastDigest 

BoxI 

PshAI 

BpiI 

BbsI 

BplI 

BplI 

Bpu10I 

Bpu10I 

Bpu1102I 

BlpI 

BseDI 

BsaJI 

BseGI 

BseGI 

BseJI 

BsaBI 

BseLI 

BslI 

BseMI 

BsrDI 

BseSI 

Bme1580I 

BseXI 

BseXI 

BshNI 

BanI 

Bsp120I 

Bsp120I 

Bsp143I 

Sau3AI 

BspLI 

NlaIV 

BspOI 

BmtI 


5’...GAm5C G(N) 3GTC...3’ 

3’...CT Gm5C(N) 3CAG...5’ 

5’...GAm5C GNNm5C GTC...3’ 

3’...CT Gm5CNN Gm5CAG...5’ 

5’...GAC(N) 4GTm5C G...3’ 

3’...CTG(N) 4CA Gm5C...5’ 

5’...GAAGAm5C G...3’ 

3’...CTTCT Gm5C...5’ 

5’...m5C GAAGAC...3’ 

3’... Gm5CTTCTG...5’ 

5’...m5C GAG(N) 5CTm5C G...3’ 

3’... Gm5CTC(N) 5GA Gm5C...5’ 

5’...CCTNAGm5C G...3’ 

3’...GGANTC Gm5C...5’ 

5’...m5C GCTNAGm5C G...3’ 

3’... Gm5CGANTC Gm5C...5’ 

5’...Cm5C Gm5C GG...3’ 

3’...G Gm5C Gm5CC...5’ 

5’...m5C GGATG...3’ 

3’... Gm5CCTAC...5’ 

5’...GAT(N) 4ATm5C G...3’ 

3’...CTA(N) 4TA Gm5C...5’ 

5’...m5C GAT(N) 4ATm5C G...3’ 

3’... Gm5CTA(N) 4TA Gm5C...5’ 

5’...Cm5C G(N) 6GG...3’ 

3’...G Gm5C(N) 6CC...5’ 

5’...Cm5C G(N) 5m5C GG...3’ 

3’...G Gm5C(N) 5 Gm5CC...5’ 

5’...m5C GCAATG...3’ 

3’... Gm5CGTTAC...5’ 

5’...m5C GKGCMm5C G...3’ 

3’... Gm5CMCGK Gm5C...5’ 

5’...m5C GCAGm5C G...3’ 

3’... Gm5CGTC Gm5C...5’ 

5’...GGYRCm5C G...3’ 

3’...CCRYG Gm5C...5’ 

5’...m5C GGYRCm5C G...3’ 

3’... Gm5CCRYG Gm5C...5’ 

5’...GGm5C GCC...3’ 

3’...CC Gm5CGG...5’ 

5’...GGGCCm5C G...3’ 

3’...CCCGG Gm5C...5’ 

5’...GATm5C G...3’ 

3’...CTA Gm5C...5’ 

5’...GGNNCm5C G...3’ 

3’...CCNNG Gm5C...5’ 

5’...m5C GGNNCm5C G...3’ 

3’... Gm5CCNNG Gm5C...5’ 

5’...GCTAGm5C G...3’ 

3’...CGATC Gm5C...5’ 

5’...m5C GCTAGm5C G...3’ 

3’... Gm5CGATC Gm5C...5’ 

? 

Conventional 

FastDigest 

BspPI 

Bst1107I 

BstZ17I 

BsuRI 

HaeIII 

BveI 

BspMI 

Cfr13I 

Sau96I 

Csp6I 

Csp6I 

DpnI 

DpnI 

Eam1104I 

EarI 

Eam1105I 

Eam1105I 

Ecl136II 

Ecl136II 

Eco24I 

Eco31I 

Eco31I 

Eco32I 

EcoRV 

Eco47I 

AvaII 

Eco91I 

Eco91I 

EcoRI 

EcoRI 

FaqI 

BsmFI 

FokI 


5’...GGATm5C G...3’ 

3’...CCTA Gm5C...5’ 

5’...m5C GGATC...3’ 

3’... Gm5CCTAG...5’ 

5’...GTATAm5C G...3’ 

3’...CATAT Gm5C...5’ 

5’...m5C GTATAm5C G...3’ 

3’... Gm5CATAT Gm5C...5’ 

5’...m5C GGCm5C G...3’ 

3’... Gm5CCG Gm5C...5’ 

5’...ACCTGm5C G...3’ 

3’...TGGAC Gm5C...5’ 

5’...GGNCm5C G...3’ 

3’...CCNG Gm5C...5’ 

5’...m5C GTAm5C G...3’ 

3’... Gm5CAT Gm5C...5’ 

5’...Gm6A Tm5C G...3’ 

3’...C Tm6A Gm5C...5’ 

5’...CTCTTm5C G...3’ 

3’...GAGAA Gm5C...5’ 

5’...GAm5C G(N) 3m5C GTC...3’ 

3’...CT Gm5C(N) 3 Gm5CAG...5’ 

5’...GAC(N) 5GTm5C G...3’ 

3’...CTG(N) 5CA Gm5C...5’ 

5’...m5C GAC(N) 5GTm5C G...3’ 

3’... Gm5CTG(N) 5CA Gm5C...5’ 

5’...GAGCTm5C G...3’ 

3’...CTCGA Gm5C...5’ 

5’...m5C GAGCTm5C G...3’ 

3’... Gm5CTCGA Gm5C...5’ 

5’...m5C GRGCYm5C G...3’ 

3’... Gm5CYCGR Gm5C...5’ 

5’...GGTCTm5C G...3’ 

3’...CCAGA Gm5C...5’ 

5’...m5C GGTCTC...3’ 

3’... Gm5CCAGAG...5’ 

5’...m5C GATATm5C G...3’ 

3’... Gm5CTATA Gm5C...5’ 

5’...GGWCm5C G...3’ 

3’...CCWG Gm5C...5’ 

5’...m5C GGTNACm5C G...3’ 

3’... Gm5CCANTG Gm5C...5’ 

5’...m5C GAATTC...3’ 

3’... Gm5CTTAAG...5’ 

5’...m5C GAATTm5C G...3’ 

3’... Gm5CTTAA Gm5C...5’ 

5’...GGGAm5C G...3’ 

3’...CCCT Gm5C...5’ 

5’...m5C GGGAC...3’ 

3’... Gm5CCCTG...5’ 

5’...m5C GGATG...3’ 

3’... Gm5CCTAC...5’’ 

www.thermoscientific.com/onebio 177 

?

178 

Table 1.18. CpG partially overlaps the recognition site. 

Conventional 

FastDigest 

HincII 

HincII 

HinfI 

HinfI 

HphI 

Hpy8I 

Hpy8I 

HpyF10VI 

HpyF10VI 

KpnI 

KpnI 

KspAI 

HpaI 

LguI 

SapI 

Lsp1109I 

BbvI 

LweI 

SfaNI 

MbiI 

BsrBI 

MboI 

MboI 

MboII 

MboII 

MnlI 

MnlI 

MssI 

MssI 

Mva1269I 

Mva1269I 

NheI 

NheI 



5’...GTYRAm5C G...3’ 

3’...CARYT Gm5C...5’ 

5’...m5C GTYRAm5C G...3’ 

3’... Gm5CARYT Gm5C...5’ 

5’...GTm5C GAC...3’ 

3’...CA Gm5CTG...5’ 

5’...GANTm5C G...3’ 

3’...CTNA Gm5C...5’ 

5’...m5C GANTm5C G...3’ 

3’... Gm5CTNA Gm5C...5’ 

5’...m5C GGTGA...3’ 

3’... Gm5CCACT...5’ 

5’...GTNNAm5C G...3’ 

3’...CANNT Gm5C...5’ 

5’...m5C GTNNAm5C G...3’ 

3’... Gm5CANNT Gm5C...5’ 

5’...GC(N) 7Gm5C G...3’ 

3’...CG(N) 7C Gm5C...5’ 

5’...m5C GC(N) 7Gm5C G...3’ 

3’... Gm5CG(N) 7C Gm5C...5’ 

5’...Gm5C G(N) 6GC...3’ 

3’...C Gm5C(N) 6CG...5’ 

5’...Gm5C G(N) 5m5C GC...3’ 

3’...C Gm5C(N) 5 Gm5CG...5’ 

5’...m5C GGTACm5C G...3’ 

3’... Gm5CCATG Gm5C...5’ 

5’...GTTAAm5C G...3’ 

3’...CAATT Gm5C...5’ 

5’...m5C GTTAAm5C G...3’ 

3’... Gm5CAATT Gm5C...5’ 

5’...m5C GCTCTTC...3’ 

3’... Gm5CGAGAAG...5’ 

5’...GCTCTTm5C G...3’ 

3’...CGAGAA Gm5C...5’ 

5’...m5C GCAGm5C G...3’ 

3’... Gm5CGTC Gm5C...5’ 

5’...GCATm5C G...3’ 

3’...CGTA Gm5C...5’ 

5’...m5C GAGCGG...3’ 

3’... Gm5CTCGCC...5’ 

5’...m5C GATm5C G...3’ 

3’... Gm5CTA Gm5C...5’ 

5’...m5C GAAGA...3’ 

3’... Gm5CTTCT...5’ 

5’...CCTm5C G...3’ 

3’...GGA Gm5C...5’ 

5’...GTTTAAAm5C G...3’ 

3’...CAAATTT Gm5C...5’ 

5’...m5C GTTTAAAm5C G...3’ 

3’... Gm5CAAATTT Gm5C...5’ 

5’...GAATGm5C G...3’ 

3’...CTTAC Gm5C...5’ 

5’...m5C GAATGC...3’ 

3’... Gm5CTTACG...5’ 

5’...GCTAGm5C G...3’ 

3’...CGATC Gm5C...5’ 

5’...m5C GCTAGm5C G...3’ 

3’... Gm5CGATC Gm5C...5’ 

? 

? 

Conventional 

FastDigest 

NmuCI 

NmuCI 

OliI 

AleI 

PaeI 

SphI 

PdmI 

PdmI 

PfeI 

TfiI 

PfoI 

PfoI 

PspFI 

PsuI 

PsuI 

PsyI 

PsyI 

RsaI 

RsaI 

RseI 

MslI 

SacI 

SacI 

SanDI 

SatI 

Fnu4HI 

SchI 

MlyI 

SduI 

Bsp1286I 

SfaNI 

SfiI 

SfiI 


5’...GTCAm5C G...3’ 

3’...CAGT Gm5C...5’ 

5’...m5C GTCAm5C G...3’ 

3’... Gm5CAGT Gm5C...5’ 

5’...CAm5C GNNNGTG...3’ 

3’...GT Gm5CNNNCAC...5’ 

5’...CAm5C GNNm5C GTG...3’ 

3’...GT Gm5CNN Gm5CAC...5’ 

5’...m5C GCATGm5C G...3’ 

3’... Gm5CGTAC Gm5C...5’ 

5’...GAA(N) 4TTm5C G...3’ 

3’...CTT(N) 4AA Gm5C...5’ 

5’...m5C GAA(N) 4TTm5C G...3’ 

3’... Gm5CTT(N) 4AA Gm5C...5’ 

5’...GAWTm5C G...3’ 

3’...CTWA Gm5C...5’ 

5’...TCm5C GGGA...3’ 

3’...AG Gm5CCCT...5’ 

5’...CCCAGm5C G...3’ 

3’...GGGTC Gm5C...5’ 

5’...m5C GGATCm5C G...3’ 

3’... Gm5CCTAG Gm5C...5’ 

5’...GAm5C GNNGTm5C G...3’ 

3’...CT Gm5CNNCA Gm5C...5’ 

5’...GTAm5C G...3’ 

3’...CAT Gm5C...5’ 

5’...m5C GTAm5C G...3’ 

3’... Gm5CAT Gm5C...5’ 

5’...CAm5C GNNNRTG...3’ 

3’...GT Gm5CNNNYAC...5’ 

5’...m5C GAGCTm5C G...3’ 

3’... Gm5CTCGA Gm5C...5’ 

5’...GGGWCCm5C G...3’ 

3’...CCCWGG Gm5C...5’ 

5’...m5C GGGWCCm5C G...3’ 

3’... Gm5CCWGG Gm5C...5’ 

5’...Gm5C GGC...3’ 

3’...C Gm5CCG...5’ 

5’...GCNGm5C G...3’ 

3’...CGNC Gm5C...5’ 

5’...GAGTm5C G...3’ 

3’...CTCA Gm5C...5’ 

5’...m5C GAGTC...3’ 

3’... Gm5CTCAG...5’ 

5’...m5C GDGCHm5C G...3’ 

3’... Gm5CHCGD Gm5C...5’ 

5’...m5C GCATC...3’ 

3’... Gm5CGTAG...5’ 

5’...GCATm5C G...3’ 

3’...CGTA Gm5C...5’ 

5’...m5C GGCC(N) 5GGCm5C G...3’ 

3’... Gm5CCGG(N) 5CCG Gm5C...5’ 

5’...GGCm5C G(N) 4GGCC...3’ 

3’...CCG Gm5C(N) 4CCGG...5’ 

5’...GGCm5C G(N) 3m5C GGCC...3’ 

3’...CCG Gm5C(N) 3 Gm5CCGG...5’ 

Conventional 

FastDigest 

SgeI** 

SmoI 

TaaI 

TaaI 

XapI 

XapI 

XceI 

NspI 

XmiI 

AccI 

Note 


** – SgeI cleaves only methylated DNA. 


m5C = 5-methylcytosine. 




5’...m5C GNG...3’ 

3’... Gm5CNC...5’ 

5’...m5C Gm5C G...3’ 

3’... Gm5C Gm5C...5’ 

5’...CTm5C GAG...3’ 

3’...GA Gm5CTC...5’ 

5’...Am5C GGT...3’ 

3’...T Gm5CCA...5’ 

5’...m5C GAATTm5C G...3’ 

3’... Gm5CTTAA Gm5C...5’ 

5’...RCATGm5C G...3’ 

3’...YGTAC Gm5C...5’ 

5’...m5C GCATGm5C G...3’ 

3’... Gm5CGTAC Gm5C...5’ 

5’...GTm5C GAC...3’ 

3’...CA Gm5CTG...5’ 

5’...GTMKAm5C G...3’ 

3’...CAKMT Gm5C...5’ 

– cleavage rate is reduced significantly 

by methylation. 

? – the sensitivity to methylation has not been 

determined. 







S = C or G; 

?

Effect of EcoKI and EcoBI Methylation on DNA Cleavage 

Methylated DNA substrates were purified from E.coli K12 or E.coli B strains. 

Table 1.19. EcoBI overlapping methylation. 

Conventional 

FastDigest 

AatII 

AatII 

AluI 

AluI 

BclI 

BclI 

BglII 

BglII 

Bpu10I 

BpuI 

Bsp143I 

Sau3AI 

EcoRI 

EcoRI 

HincII 

HincII 

HindIII 

HindIII 

HinfI 

HinfI 

HphI 

MboI 

MboI 

MboII 

MboII 

MluI 

MluI 

MnlI 

MnlI 

NdeI 

NdeI 

OliI 

AleI 

PaeI 

SphI 

PagI 

BspHI 

SacI 

SacI 

ScaI 

ScaI 

SspI 

SspI 

TasI 

Tsp509I 


5’...TGm6ACGTC(N) 4 TGCT...3’ 

3’...AC TGCAG(N) 4m6ACGA...5’ 

5’...TGm6AGCT(N) 5 TGCT...3’ 

3’...AC TCGA(N) 5m6ACGA...5’ 

5’...TGm6A TCA(N) 5 TGCT...3’ 

3’...AC Tm6AGT(N) 5m6ACGA...5’ 

5’...TGm6AGATCT(N) 3 TGCT...3’ 

3’...AC TCTAGA(N) 3m6ACGA...5’ 

5’...CCTGm6AGC(N) 6 TGCT...3’ 

3’...GGAC TCG(N) 6m6ACGA...5’ 

5’...TGm6ATC(N) 6 TGCT...3’ 

3’...AC TAG(N) 6m6ACGA...5’ 

5’...TGm6AATTC(N) 4 TGCT...3’ 

3’...AC TTAAG(N) 4m6ACGA...5’ 

5’...GTTGm6AC(N) 7 TGCT...3’ 

3’...CAAC TG(N) 7m6ACGA...5’ 

5’...TGm6AAGCTT(N) 3 TGCT...3’ 

3’...AC TTCGAA(N) 3m6ACGA...5’ 

5’...TGm6ANTC(N) 5 TGCT...3’ 

3’...AC TNAG(N) 5m6ACGA...5’ 

5’...GGTGm6A(N) 8 TGCT...3’ 

3’...CCAC T(N) 8m6ACGA...5’ 

5’...TGm6ATC(N) 6 TGCT...3’ 

3’...AC TAG(N) 6m6ACGA...5’ 

5’...TGm6AAGA(N) 5 TGCT...3’ 

3’...AC TTCT(N) 5m6ACGA...5’ 

5’...TGm6ACGCGT(N) 3 TGCT...3’ 

3’...AC TGCGCA(N) 3m6ACGA...5’ 

5’...TGm6AGG(N) 6 TGCT...3’ 

3’...AC TCC(N) 6m6ACGA...5’ 

5’...TGm6A(N) 4CATA TGCT...3’ 

3’...AC T(N) 4GTATm6ACGA...5’ 

5’...TGm6A(N) 4CACG TGCT...3’ 

3’...AC T(N) 4GTGCm6ACGA...5’ 

5’...TGm6A(N) 5GCA TGCT...3’ 

3’...AC T(N) 5CGTm6ACGA...5’ 

5’...TCATGm6A(N) 8 TGCT...3’ 

3’...AGTAC T(N) 8m6ACGA...5’ 

5’...TGm6AGCTC(N) 4 TGCT...3’ 

3’...AC TCGAG(N) 4m6ACGA...5’ 

5’...TGm6AGTACT(N) 3 TGCT...3’ 

3’...AC TCATGA(N) 3m6ACGA...5’ 

5’...TGm6AATATT(N) 3 TGCT...3’ 

3’...AC TTATAA(N) 3m6ACGA...5’ 

5’...TGm6AATT(N) 5 TGCT...3’ 

3’...AC TTAA(N) 5m6ACGA...5’ 

Table 1.20. EcoKI overlapping methylation. 

Conventional 

FastDigest 

Alw21I 

Alw21I 

Alw44I 

ApaLI 

BseSI 

Bme1580I 

DraI 

DraI 

HincII 

HincII 

KspAI 

HpaI 

MluI 

MluI 

MssI 

MssI 

NsbI 

FspI 

OliI 

AleI 

RseI 

MslI 

Tru1I 

Tru1I 

Note 





5’...Am6AC(N) 6G TGCWC...3’ 

3’...T TG(N) 6Cm6ACGWG...5’ 

5’...Am6AC(N) 6G TGCAC...3’ 

3’...T TG(N) 6Cm6ACGTG...5’ 

5’...Am6AC(N) 6G TGCMC...3’ 

3’...T TG(N) 6Cm6ACGKG...5’ 

5’...TTTAAm6AC(N) 6G TGC...3’ 

3’...AAATT TG(N) 6Cm6ACG...5’ 

5’...GTYAm6AC(N) 6G TGC...3’ 

3’...CART TG(N) 6Cm6ACG...5’ 

5’...GTTAm6AC(N) 6G TGC...3’ 

3’...CAAT TG(N) 6Cm6ACG...5’ 

5’...Am6ACGCGTNNG TGC...3’ 

3’...T TGCGCANNCm6ACG...5’ 

5’...GTTTAAm6AC(N) 6G TGC...3’ 

3’...CAAATT TG(N) 6Cm6ACG...5’ 

5’...Am6AC(N) 6G TGCGCA...3’ 

3’...T TG(N) 6Cm6ACGCGT...5’ 

5’...Am6ACAC(N) 4G TGC...3’ 

3’...T TGTG(N) 4Cm6ACG...5’ 

5’...GCm6ACNNNNRTG TT...3’ 

3’...CG TGNNNNYACm6AT...5’ 

5’...TTAm6AC(N) 6G TGC...3’ 

3’...AAT TG(N) 6Cm6ACG...5’ 

– cleavage rate is reduced significantly by 

methylation. 







S = C or G; 

Recognition sequences of the following 

endonucleases may also overlap with DNA 

sequences methylated by EcoBI and EcoKI. 

The following conventional restriction enzymes 

and the respective FastDigest enzymes have not 

been tested for sensitivity to EcoBI methylation: 

AarI, AdeI, AjiI, Alw21I, BauI, BcuI, BglII, BoxI, 

 

BseXI, BshTI, Bsp143II, BspPI, Bsu15I, BveI, 

CaiI, Cfr10I, CseI, DpnI, Eam1104I, Ecl136II, 

Eco24I, Eco31I, Eco32I, Eco47III, Eco57I, 

Eco72I, Eco81I, Eco91I, Eco147I, EcoO109I, 

Esp3I, FspAI, FokI*, HaeII*, HindIII, Hin1I, 

Hin1II, Hpy8I, HpyF3I, LguI, Lsp1109I, LweI, 

MbiI, Mph1103I, MunI, Mva1269I, NdeI, 

NmuCI, PdmI, PfeI, Ppu21I, PscI, Psp5II, 

Psp1406I, PsuI, PsyI, PvuII, RseI, SchI, SduI, 

SexAI*, SfaNI*, SmiI, SmoI, SspI, TaaI, TaiI, 

TatI, TscAI, VspI, XagI, XapI and XceI. 

The following conventional restriction enzymes 

and the respective FastDigest enzymes have not 

been tested for sensitivity to EcoKI methylation: 

AanI, AdeI, AjiI, BauI, BcuI, BseNI, BshNI, 

BshTI, Bsp119I, BveI, Cfr10I, Eco72I, Eco91I, 

FspAI, Hpy8I, MseI*, PacI, PdmI, Ppu21I, PscI, 

Psp1406I, SexAI*, SduI, TaaI, TaiI, TscAI 

and XceI. 

* enzymes are available in FastDigest format only. 


180 

Newly Generated Cleavage Sites 

Recognition Sites Resulting from Ligation of Blunt DNA Ends 

Note 

* Restriction enzymes that have degenerate recognition sequences 

 

Be aware that these restriction enzymes will cleave sequences in 

addition to the one listed. 

enzymes are shown in purple. 

Thermo Scientific conventional enzymes are shown in orange. 

Table 1.21. Newly generated recognition sites resulting from ligation of blunt DNA ends. 

First restriction 

enzyme 

Second restriction enzyme 

Restriction enzymes that cleave the newly generated 

recognition sequence 

Bst1107I /FastDigest BstZ17I 

DraI/FastDigest DraI Eco32I /FastDigest EcoRV HincII 

FaiI 

AanI / 

FastDigest PsiI 

 

/FastDigest HincII KspAI /FastDigest HpaI MssI / 

FastDigest MssI RsaI/FastDigest RsaI ScaI/FastDigest ScaI 

SmiI /FastDigest SwaI 

Eco147I /FastDigest StuI 

Eco47III /FastDigest AfeI 

FastDigest MseI , Tru1I /FastDigest Tru1I 

SetI 

AluI/FastDigest AluI 

EheI /FastDigest EheI 

SspI/FastDigest SspI 

FastDigest MseI , TasI /FastDigest 

Tsp509I , Tru1I /FastDigest Tru1I 

AluI/FastDigest AluI PvuII/FastDigest PvuII SetI 

Ecl136II /FastDigest Ecl136II MbiI /FastDigest BsrBI MnlI/FastDigest MnlI, SetI 

Eco105I /FastDigest SnaBI Ppu21I /FastDigest BsaAI * MaeII, Ppu21I /FastDigest BsaAI , SetI, TaiI 

 

/FastDigest TaiI 

AjiI * 

 

Eco72I /FastDigest PmlI Ppu21I /FastDigest BsaAI * 

 

Eco72I /FastDigest PmlI , MaeII, Ppu21I 

/FastDigest BsaAI , SetI, TaiI / 


TscAI /FastDigest TspRI 

MbiI /FastDigest BsrBI HpaII/FastDigest HpaII, MspI /FastDigest MspI 

PdiI /FastDigest NaeI BceAI 

AjiI , MaeII, SetI, TaiI /FastDigest TaiI 


DpnI/FastDigest DpnI HinfI/FastDigest HinfI 

Ecl136II /FastDigest Ecl136II MbiI /FastDigest BsrBI 

Eco105I /FastDigest SnaBI Eco72I /FastDigest PmlI 

MnlI/FastDigest MnlI, SetI 

Ppu21I /FastDigest BsaAI Ppu21I /FastDigest BsaAI 

 

MaeII, SetI, TaiI /FastDigest TaiI 

AjiI * 

 

Eco47III /FastDigest AfeI FspAI/FastDigest FspAI FspAI/ 

FastDigest FspAI NsbI /FastDigest FspI 

CseI /FastDigest HgaI 


CseI /FastDigest HgaI , Hin1I / 




 

AjiI AjiI Eco105I /FastDigest SnaBI 

AatII/FastDigest AatII, Hin1I /FastDigest BsaHI 

, MaeII, SetI, TaiI /FastDigest TaiI, ZraI 

Eco72I /FastDigest PmlI Ppu21I /FastDigest BsaAI 

Ppu21I /FastDigest BsaAI 

SetI 


BsuRI /FastDigest HaeIII Eco147I /FastDigest StuI 

Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI 

AluI/FastDigest AluI Eco47III /FastDigest AfeI MlsI /FastDigest 

 

MscI 

Ecl136II /FastDigest Ecl136II MbiI /FastDigest BsrBI 

PvuII/FastDigest PvuII 


Eco32I /FastDigest EcoRV 

Bsp143I /FastDigest Sau3AI , DpnI/ 

FastDigest DpnI, MboI/FastDigest MboI 

EheI /FastDigest EheI BsuRI /FastDigest HaeIII 

Bst1107I /FastDigest BstZ17I Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI 

Bsh1236I / 




FastDigest Bsh1236I Eco47III /FastDigest AfeI 

 


Bsp68I /FastDigest NruI MbiI /FastDigest BsrBI 

 

Bsh1236I /FastDigest Bsh1236I 

 








S = C or G;



enzyme 




Bsh1236I /FastDigest Bsh1236I MbiI /FastDigest BsrBI 

 



DraI/FastDigest DraI HincII /FastDigest HincII KspAI / 


Bsp68I / 

FastDigest HpaI MssI /FastDigest MssI RsaI/FastDigest RsaI 

TaqI/FastDigest TaqI 

ScaI/FastDigest ScaI SmiI /FastDigest SwaI SspI/ 

FastDigest NruI FastDigest SspI 

 



FastDigest DpnI, MboI/FastDigest MboI, TaqI/ 




HincII /FastDigest HincII Hpy188I 

AanI /FastDigest PsiI 

AluI/FastDigest AluI Bsh1236I /FastDigest Bsh1236I Bsp68I / 

FastDigest NruI BsuRI /FastDigest HaeIII 

FaiI 

Ecl136II /FastDigest Ecl136II MbiI / 

FastDigest BsrBI MbiI /FastDigest BsrBI MlsI / 

FastDigest MscI PvuII/FastDigest PvuII 

 


Bst1107I / 

FastDigest BstZ17I 

 



Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI, 

SetI 



HincII /FastDigest HincII KspAI /FastDigest HpaI Hpy8I /FastDigest Hpy8I 

HincII /FastDigest HincII 

Hpy8I /FastDigest Hpy8I, XmiI /FastDigest 

AccI 

RsaI/FastDigest RsaI ScaI/FastDigest ScaI MaeIII 


AluI/FastDigest AluI Ecl136II /FastDigest Ecl136II 

TasI /FastDigest Tsp509I 

Eco47III /FastDigest AfeI MbiI /FastDigest BsrBI 


 


BsuRI / 

Eco147I /FastDigest StuI MlsI /FastDigest MscI 

BsuRI /FastDigest HaeIII 

 

FastDigest HaeIII 

 


Bsp143I /FastDigest Sau3AI , BspPI 

, DpnI/FastDigest DpnI, MboI/FastDigest MboI 


BmgT120I, BsuRI /FastDigest HaeIII , Cfr13I 

/FastDigest Sau96I 

FspAI/FastDigest FspAI 

BseSI /FastDigest Bme1580I , SduI 

/FastDigest Bsp1286I 



FaqI /FastDigest BsmFI 



SetI 

AluI/FastDigest AluI Ecl136II /FastDigest Ecl136II MbiI / 

FastDigest BsrBI PvuII/FastDigest PvuII 



BsuRI /FastDigest HaeIII Eco147I /FastDigest StuI 

Eco47III /FastDigest AfeI MlsI /FastDigest MscI 

 





AluI/FastDigest AluI Ecl136II /FastDigest Ecl136II Eco47III / 


FastDigest AfeI MbiI /FastDigest BsrBI 


 


BsuRI /FastDigest HaeIII Eco147I /FastDigest StuI 

MlsI /FastDigest MscI 


 










HincII /FastDigest HincII FaqI /FastDigest BsmFI 

 


182 



enzyme 




AjiI 

HinfI/FastDigest HinfI, PleI, SchI /FastDigest MlyI 

 

Ecl136II /FastDigest Ecl136II MbiI /FastDigest BsrBI HinfI/FastDigest HinfI 

Eco147I /FastDigest StuI SetI 

DpnI/FastDigest Eco32I /FastDigest EcoRV HinfI/FastDigest HinfI, PfeI /FastDigest TfiI 

DpnI Eco47III /FastDigest AfeI AluI/FastDigest AluI 



Alw21I /FastDigest Alw21I, SduI / 


SspI/FastDigest SspI TasI /FastDigest Tsp509I 

AanI /FastDigest PsiI HincII /FastDigest HincII KspAI / 



Bsp68I /FastDigest NruI 

DraI/FastDigest DraI 

FspAI/FastDigest FspAI FspAI/FastDigest FspAI NsbI / 

 



HpyCH4V 

MssI /FastDigest MssI SmiI /FastDigest SwaI 


DraI/FastDigest DraI, FastDigest MseI , Tru1I 

/FastDigest Tru1I 



SetI 

AluI/FastDigest AluI PvuII/FastDigest PvuII AluI/FastDigest AluI 




Eco47III /FastDigest AfeI MlsI /FastDigest 

Ecl136II / MscI 


 

DpnI/FastDigest DpnI 


 





AluI/FastDigest AluI, Alw21I /FastDigest 

MbiI /FastDigest BsrBI 

Alw21I Ecl136II /FastDigest Ecl136II, 

Eco24I , SacI/FastDigest SacI, SduI / 

FastDigest Bsp1286I , SetI 

AjiI MaeII, SetI, TaiI /FastDigest TaiI 

AjiI Eco72I /FastDigest PmlI Ppu21I / MaeII, Ppu21I /FastDigest BsaAI , SetI, TaiI 



Eco105I / 

FastDigest SnaBI 

 





Eco105I /FastDigest SnaBI , MaeII, 


Ppu21I /FastDigest BsaAI , SetI, TaiI 


AanI /FastDigest PsiI DpnI/FastDigest DpnI SetI 




 


Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI, 

SetI 

Eco147I / 

FastDigest StuI 

BsuRI /FastDigest HaeIII MlsI /FastDigest MscI 

 


 










HincII /FastDigest HincII FaqI /FastDigest BsmFI 




enzyme 





AluI/FastDigest AluI Bsh1236I /FastDigest Bsh1236I BsuRI / 

FastDigest HaeIII Ecl136II /FastDigest 


Ecl136II Eco147I /FastDigest StuI MbiI Bsp143I /FastDigest Sau3AI , DpnI/ 

/FastDigest BsrBI MbiI /FastDigest BsrBI MlsI FastDigest DpnI, MboI/FastDigest MboI 

Eco32I / /FastDigest MscI PvuII/FastDigest 

FastDigest EcoRV PvuII 

 



FastDigest DpnI, MboI/FastDigest MboI, TaqI/ 


DpnI/FastDigest DpnI HinfI/FastDigest HinfI, PfeI /FastDigest TfiI 

FspAI/FastDigest FspAI NsbI /FastDigest FspI HpyCH4V 

FspAI/FastDigest FspAI HpyCH4V, Mph1103I /FastDigest NsiI 

AanI /FastDigest PsiI Bst1107I /FastDigest BstZ17I 




FastDigest NruI BsuRI /FastDigest HaeIII 

Eco147I /FastDigest StuI MlsI / 

FastDigest MscI PvuII/FastDigest PvuII 

 

Ecl136II /FastDigest Ecl136II MbiI /FastDigest BsrBI MnlI/FastDigest MnlI 

Eco47III / Eco32I /FastDigest EcoRV LweI /FastDigest SfaNI 

FastDigest AfeI 

 


FastDigest HaeII , HhaI/FastDigest HhaI, Hin6I 

/FastDigest HinP1I 

FspAI/FastDigest FspAI FspAI/FastDigest FspAI NsbI / HhaI/FastDigest HhaI, Hin6I /FastDigest HinP1I 


 


HpaII/FastDigest HpaII, MspI /FastDigest 

MspI 

PdiI /FastDigest NaeI 

SatI /FastDigest Fnu4HI , TauI/FastDigest 

TauI 

Cac8I 

AjiI AjiI , MaeII, SetI, TaiI /FastDigest TaiI 


AjiI Ppu21I /FastDigest BsaAI 



Eco72I / AluI/FastDigest AluI PvuII/FastDigest PvuII SetI 

FastDigest PmlI Ecl136II /FastDigest Ecl136II MbiI /FastDigest BsrBI MnlI/FastDigest MnlI, SetI 

Eco105I /FastDigest SnaBI Ppu21I /FastDigest BsaAI * MaeII, Ppu21I /FastDigest BsaAI , SetI, TaiI 

 







 

AjiI 


Hin1I /FastDigest BsaHI 

FastDigest NruI PvuII/ 

FastDigest PvuII 


BsuRI /FastDigest HaeIII Eco147I /FastDigest StuI BmgT120I, BsuRI /FastDigest HaeIII , Cfr13I 



EheI / 


 




BsuRI /FastDigest HaeIII MnlI/ 


LweI /FastDigest SfaNI 

FastDigest HaeII , HhaI/FastDigest HhaI, Hin6I 


FspAI/FastDigest FspAI FspAI/FastDigest FspAI NsbI / HhaI/FastDigest HhaI, Hin6I /FastDigest HinP1I 


 


BsuRI /FastDigest HaeIII HpaII/ 

FastDigest HpaII, MspI /FastDigest MspI 



TauI 

Cac8I 


 

FaiI 

 

AjiI Eco72I /FastDigest PmlI 

Ppu21I /FastDigest BsaAI PvuII/FastDigest PvuII 



SetI 

Eco47III /FastDigest AfeI AluI/FastDigest AluI 


SspI/FastDigest SspI TasI /FastDigest Tsp509I 

 


184 



enzyme 





 

FaiI 

 



SetI 






FastDigest HpaI MssI /FastDigest MssI RsaI/FastDigest RsaI HpyCH4VI 

ScaI/FastDigest ScaI SmiI /FastDigest SwaI 



LweI /FastDigest SfaNI , HpyCH4V, Mph1103I 

/FastDigest NsiI 

FspAI/FastDigest 

FspAI 

Eco47III /FastDigest AfeI EheI /FastDigest EheI 


HhaI/FastDigest HhaI, Hin6I /FastDigest HinP1I 

 

HpaII/FastDigest HpaII, MspI /FastDigest MspI 

NsbI /FastDigest FspI 


, NsbI /FastDigest FspI 



TauI 

Cac8I 

SspI/FastDigest SspI HpyCH4V, Mph1103I /FastDigest NsiI 

BsuRI /FastDigest HaeIII Eco147I /FastDigest StuI BseSI /FastDigest Bme1580I , SduI 





Alw21I /FastDigest Alw21I, SduI / 


FastDigest HpaI MssI /FastDigest MssI SmiI / 

FastDigest SwaI SspI/FastDigest SspI 

HpyCH4V 


Eco32I /FastDigest EcoRV LweI /FastDigest SfaNI , HpyCH4V 

FspAI/FastDigest 

FspAI 




 


NsbI /FastDigest FspI 





TauI 

Alw21I /FastDigest Alw21I, Alw44I / 

RsaI/FastDigest RsaI ScaI/FastDigest ScaI 

FastDigest ApaLI , BseSI /FastDigest 

Bme1580I , Hpy8I /FastDigest Hpy8I, 

HpyCH4V, SduI /FastDigest Bsp1286I 

Cac8I 

Bsp68I /FastDigest NruI Hpy188I 


Hpy8I /FastDigest Hpy8I, XmiI /FastDigest 

AccI 

HincII / DpnI/FastDigest DpnI Alw26I /FastDigest Alw26I 

FastDigest HincII* Ecl136II /FastDigest Ecl136II MbiI /FastDigest BsrBI MnlI/FastDigest MnlI 

 

KspAI /FastDigest HpaI 

HincII /FastDigest HincII, Hpy8I /FastDigest 

Hpy8I 



AanI /FastDigest PsiI Bsp68I /FastDigest NruI DraI/ 

MaeIII, NmuCI /FastDigest NmuCI 

FastDigest DraI MssI /FastDigest MssI SmiI /FastDigest SwaI FastDigest MseI , Tru1I /FastDigest Tru1I 

 

Bsp68I /FastDigest NruI TaqI/FastDigest TaqI 

HincII / Bst1107I /FastDigest BstZ17I Hpy8I /FastDigest Hpy8I 

FastDigest HincII* 

 



HpyCH4V 



Hpy8I, KspAI /FastDigest HpaI , FastDigest 

MseI , Tru1I /FastDigest Tru1I 

RsaI/FastDigest RsaI ScaI/FastDigest ScaI MaeIII 


 






 




enzyme 




AanI /FastDigest PsiI DraI/FastDigest DraI MssI /FastDigest 

MssI SmiI /FastDigest SwaI 



Bst1107I /FastDigest BstZ17I Hpy8I /FastDigest Hpy8I 

KspAI / 

FastDigest HpaI 

 




HpyCH4V 


Hpy8I, KspAI /FastDigest HpaI , FastDigest 

MseI , Tru1I /FastDigest Tru1I 



Hpy8I 



Eco72I /FastDigest PmlI FspAI/FastDigest FspAI* 

MaeIII 

FspAI/FastDigest FspAI HincII /FastDigest HincII 

NsbI /FastDigest FspI Ppu21I /FastDigest BsaAI 


AluI/FastDigest AluI BsuRI /FastDigest HaeIII 




SsiI /FastDigest AciI 

Bsh1236I /FastDigest Bsh1236I Bsp68I /FastDigest NruI 

Bsh1236I /FastDigest Bsh1236I, SsiI / 



Ecl136II /FastDigest Ecl136II 

MbiI /FastDigest BsrBI , SsiI /FastDigest 

AciI 

MbiI / 

FastDigest BsrBI 

 






MspI 




BseDI /FastDigest BsaJI , Bsh1236I / 

 

FastDigest Bsh1236I, BtgI, Cfr42I , MspA1I, SsiI 

/FastDigest AciI 

PvuII/FastDigest PvuII MspA1I, SsiI /FastDigest AciI 

BcnI /FastDigest NciI , Bme1390I / 


FastDigest ScrFI , BssKI, HpaII/FastDigest 

HpaII, MspI /FastDigest MspI 


SmaI/FastDigest SmaI 


FastDigest ScrFI , BseDI /FastDigest 

BsaJI , BssKI, HpaII/FastDigest HpaII, MspI / 




SetI 

AluI/FastDigest AluI PvuII/FastDigest PvuII AluI/FastDigest AluI 




Eco47III /FastDigest AfeI MlsI /FastDigest 

MbiI / MscI 

FastDigest BsrBI 

 



 

AluI/FastDigest AluI, Alw21I /FastDigest 

Ecl136II /FastDigest Ecl136II 

Alw21I Ecl136II /FastDigest Ecl136II, 

Eco24I , SacI/FastDigest SacI, SduI / 

FastDigest Bsp1286I , SetI 





 


186 



enzyme 

MlsI / 

FastDigest MscI 

 

 

 

MssI / 


 








 



 













FaqI /FastDigest BsmFI 



SetI 

AluI/FastDigest AluI Ecl136II /FastDigest Ecl136II MbiI 


/FastDigest BsrBI 




Eco47III /FastDigest AfeI MlsI /FastDigest 

MscI 

 






MbiI /FastDigest BsrBI MspA1I 



Eco72I /FastDigest PmlI FspAI/FastDigest FspAI* 

AluI/FastDigest AluI PvuII/FastDigest 

PvuII, SetI 

FspAI/FastDigest FspAI HincII /FastDigest HincII 

NsbI /FastDigest FspI Ppu21I /FastDigest BsaAI 


AluI/FastDigest AluI BsuRI /FastDigest HaeIII 





Bsh1236I /FastDigest Bsh1236I Bsp68I /FastDigest NruI 

Bsh1236I /FastDigest Bsh1236I, SsiI / 




MbiI /FastDigest BsrBI , SsiI /FastDigest 

AciI 






MspI 




BseDI /FastDigest BsaJI , Bsh1236I / 


FastDigest Bsh1236I, BtgI, Cfr42I , MspA1I, SsiI 






PvuII/FastDigest PvuII MspA1I, SsiI /FastDigest AciI 







FastDigest MseI , Tru1I /FastDigest Tru1I, 



DraI/FastDigest DraI SmiI /FastDigest SwaI 





HpyCH4V 

RsaI/FastDigest RsaI ScaI/FastDigest ScaI Hpy8I /FastDigest Hpy8I



enzyme 

NsbI / 

FastDigest FspI 

 

PdiI / 

FastDigest NaeI 

 





FastDigest HpaI MssI /FastDigest MssI RsaI/FastDigest RsaI 

HpyCH4V 

ScaI/FastDigest ScaI SmiI /FastDigest SwaI SspI/ 

FastDigest SspI 


Eco32I /FastDigest EcoRV LweI /FastDigest SfaNI , HpyCH4V 



 

FspAI/FastDigest FspAI FspAI/FastDigest FspAI 






TauI 

Cac8I 

DpnI/FastDigest DpnI Ecl136II /FastDigest Ecl136II MbiI / 


MnlI/FastDigest MnlI 

Eco32I /FastDigest EcoRV BccI 

Eco47III /FastDigest AfeI EheI /FastDigest EheI FspAI/ 

SatI /FastDigest Fnu4HI , SsiI / 

FastDigest FspAI FspAI/FastDigest FspAI NsbI /FastDigest FspI 

FastDigest AciI , TauI/FastDigest TauI 

 

MbiI /FastDigest BsrBI SmaI/FastDigest SmaI 

 




AjiI AjiI , MaeII, SetI, TaiI /FastDigest TaiI 





Ppu21I / AluI/FastDigest AluI PvuII/FastDigest PvuII SetI 

FastDigest BsaAI Ecl136II /FastDigest Ecl136II MbiI /FastDigest BsrBI MnlI/FastDigest MnlI, SetI 

 

Eco105I /FastDigest SnaBI 

MaeII, Ppu21I /FastDigest BsaAI , SetI, TaiI 





AjiI MaeII, SetI, TaiI /FastDigest TaiI 


MaeII, Ppu21I /FastDigest BsaAI , SetI, TaiI 


Ppu21I / 

FastDigest BsaAI 

 



Eco105I /FastDigest SnaBI , MaeII, 

Eco105I /FastDigest SnaBI 

Ppu21I /FastDigest BsaAI , SetI, TaiI 





BceAI 



SetI 

AluI/FastDigest AluI Ecl136II /FastDigest Ecl136II MbiI 


/FastDigest BsrBI 




PvuII/FastDigest Eco47III /FastDigest AfeI MlsI /FastDigest 

PvuII MscI 






MbiI /FastDigest BsrBI MspA1I 

 

AluI/FastDigest AluI PvuII/FastDigest 

PvuII, SetI 

 


188 



enzyme 

RsaI/FastDigest 

RsaI 

ScaI/FastDigest 

ScaI 

SmaI/FastDigest 

SmaI 

SmiI / 

FastDigest SwaI 

 





AanI /FastDigest PsiI FastDigest MseI , Tru1I /FastDigest Tru1I 


Bst1107I /FastDigest BstZ17I HincII /FastDigest HincII* 


MaeIII 

Ecl136II /FastDigest Ecl136II MbiI /FastDigest BsrBI Alw26I /FastDigest Alw26I 







HincII /FastDigest HincII MaeIII, NmuCI /FastDigest NmuCI 

MssI /FastDigest MssI Hpy8I /FastDigest Hpy8I 

ScaI/FastDigest ScaI Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI 

AanI /FastDigest PsiI FastDigest MseI , Tru1I /FastDigest Tru1I 


Bst1107I /FastDigest BstZ17I HincII /FastDigest HincII* 


MaeIII 

Ecl136II /FastDigest Ecl136II MbiI /FastDigest BsrBI Alw26I /FastDigest Alw26I 







HincII /FastDigest HincII MaeIII, NmuCI /FastDigest NmuCI 

MssI /FastDigest MssI Hpy8I /FastDigest Hpy8I 

RsaI/FastDigest RsaI Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI 






BccI 

FastDigest FspAI FspAI/FastDigest FspAI NsbI /FastDigest FspI FauI, SsiI /FastDigest AciI 

 











FastDigest ScrFI , BmeT110I, BseDI / 

 

FastDigest BsaJI , BssKI, Cfr9I , Eco88I 

/FastDigest AvaI , HpaII/FastDigest HpaII, 

MspI /FastDigest MspI, SmaI/FastDigest SmaI 





DraI/FastDigest DraI MssI /FastDigest MssI 





HpyCH4V



enzyme 









BccI 

FastDigest FspAI FspAI/FastDigest FspAI NsbI /FastDigest FspI Cac8I, FauI, SsiI /FastDigest AciI 

 


 










FastDigest ScrFI , BmeT110I, BseDI / 


FastDigest BsaJI , BssKI, Cfr9I , Eco88I 

/FastDigest AvaI , HpaII/FastDigest HpaII, 

MspI /FastDigest MspI, SmaI/FastDigest SmaI 


FastDigest MseI , TasI /FastDigest 

Tsp509I , Tru1I /FastDigest Tru1I 

SspI/FastDigest 

SspI 


Bst1107I /FastDigest BstZ17I DpnI/FastDigest DpnI 

 




FspAI/FastDigest FspAI HpyCH4V, Mph1103I /FastDigest NsiI 

AjiI 

AjiI Eco105I /FastDigest SnaBI Eco72I / 

AatII/FastDigest AatII, Hin1I /FastDigest BsaHI 

, MaeII, SetI, TaiI /FastDigest TaiI, ZraI 

FastDigest PmlI Ppu21I /FastDigest BsaAI Ppu21I 

/FastDigest BsaAI 



 



HinfI/FastDigest HinfI 

MnlI/FastDigest MnlI, SetI 

Eco47III /FastDigest AfeI FspAI/FastDigest FspAI FspAI/ 

FastDigest FspAI NsbI /FastDigest FspI 



CseI /FastDigest HgaI , Hin1I / 





190 

Recognition Sites Resulting from Ligation of Protruding Compatible DNA Ends 

Note 


 

enzymes will cleave sequences in addition to the one listed. 



Table 1.22. Newly generated recognition sites resulting from ligation of protruding compatible DNA ends. 

First 

Second 

Restriction enzyme that cleave the newly generated recognition 

restriction enzyme restriction enzyme 

sequence 

AatII/FastDigest AatII 

 

TaiI /FastDigest TaiI MaeII, SetI, TaiI /FastDigest TaiI 

Eco88I /FastDigest AvaI SmoI BmeT110I, Eco88I /FastDigest AvaI , MnlI/FastDigest MnlI, 

XhoI/FastDigest XhoI 

SmoI , TaqI/FastDigest TaqI, XhoI/FastDigest XhoI 

 

 

 

BmeT110I, Eco88I /FastDigest AvaI , MnlI/FastDigest MnlI, 

PspXI, SmoI , TaqI/FastDigest TaqI, XhoI/FastDigest XhoI 

AbsI, BmeT110I, Eco88I /FastDigest AvaI , MnlI/FastDigest 

MnlI, PspXI, SmoI , TaqI/FastDigest TaqI, XhoI/FastDigest XhoI 

SalI/FastDigest SalI MnlI/FastDigest MnlI, TaqI/FastDigest TaqI 

SgrDI Hpy99I, MnlI/FastDigest MnlI, TaqI/FastDigest TaqI 

Acc65I /FastDigest Acc65I, BshNI /FastDigest BanI , 

BshNI /FastDigest BanI 

BspLI /FastDigest NlaIV , Csp6I /FastDigest Csp6I, 

Acc65I /FastDigest 

KpnI/FastDigest KpnI, RsaI/FastDigest RsaI 

Acc65I Bsp1407I /FastDigest Bsp1407I Pfl23II / 

FastDigest BsiWI TatI/FastDigest TatI 

TatI/FastDigest TatI 

Eco130I /FastDigest StyI 


NcoI/FastDigest NcoI PagI /FastDigest BspHI CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII 

 

 

PscI 

AflIII, CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII , PscI , XceI 

/FastDigest NspI 


 

MauBI/FastDigest MauBI SgsI /FastDigest AscI Bsh1236I /FastDigest Bsh1236I, HhaI/FastDigest HhaI, Hin6I 

PauI /FastDigest BssHII /FastDigest HinP1I 

MluI/FastDigest MluI AflIII, Bsh1236I /FastDigest Bsh1236I, MluI/FastDigest MluI 


Alw21I /FastDigest 

SduI /FastDigest Bsp1286I 

Alw21I 

Alw21I /FastDigest Alw21I, SduI /FastDigest Bsp1286I 

 

Eco24I SacI/FastDigest SacI SduI 



Alw21I 

AluI/FastDigest AluI, Alw21I /FastDigest Alw21I Ecl136II 

/FastDigest Ecl136II, Eco24I , SacI/FastDigest SacI, SduI 

/FastDigest Bsp1286I , SetI 


BseSI /FastDigest Bme1580I SduI 



Alw21I /FastDigest Alw21I, Alw44I /FastDigest ApaLI 

, BseSI /FastDigest Bme1580I , Hpy8I / 

FastDigest Hpy8I, HpyCH4V, SduI /FastDigest Bsp1286I 

Alw21I Mph1103I /FastDigest NsiI HpyCH4V 

PstI/FastDigest PstI SdaI /FastDigest SbfI 

 

BsgI, HpyCH4V 



Alw21I 


 


ApaLI 

BfmI /FastDigest SfcI BsgI, HpyCH4V 

ApaI/FastDigest ApaI, BmgT120I, BseSI /FastDigest Bme1580I 

ApaI/FastDigest ApaI 

 

, Bsp120I /FastDigest Bsp120I, BspLI /FastDigest 

BseSI /FastDigest Bme1580I Eco24I * 

NlaIV , BsuRI /FastDigest HaeIII , Cfr13I / 


FastDigest Sau96I Eco24I , SduI /FastDigest 

Bsp1286I 

BclI/FastDigest BclI Bsp143I /FastDigest Sau3AI Bsp143I /FastDigest Sau3AI , BspPI , DpnI/ 

MboI/FastDigest MboI 


BamHI/FastDigest BamHI 

 

BglII/FastDigest BglII PsuI /FastDigest PsuI 

Bsp143I /FastDigest Sau3AI , BspPI , DpnI/ 

FastDigest DpnI, MboI/FastDigest MboI, PsuI /FastDigest PsuI 

BamHI/FastDigest BamHI, Bsp143I /FastDigest Sau3AI , 

PsuI /FastDigest PsuI 

BspLI /FastDigest NlaIV , BspPI , DpnI/FastDigest DpnI, 

MboI/FastDigest MboI, PsuI /FastDigest PsuI 

BbeI, FastDigest HaeII , BshNI /FastDigest BanI , 

 


BspLI /FastDigest NlaIV , EheI /FastDigest EheI, HhaI/ 

FastDigest HhaI, Hin1I /FastDigest BsaHI , Hin6I / 

FastDigest HinP1I , NarI, SspDI 


FastDigest HaeII , HhaI/FastDigest HhaI, Hin6I /FastDigest 

HinP1I 








S = C or G;


First 


BclI/FastDigest BclI 

 

BcuI /FastDigest SpeI 

 

BfmI /FastDigest SfcI 

 

BglII/FastDigest BglII 

 

 

 

 

 

BseSI / 

FastDigest Bme1580I 

 

BseSI / 


 

BseSI / 


 

BseSI / 


 

Second 


BamHI/FastDigest BamHI BglII/FastDigest BglII 

Bsp143I /FastDigest Sau3AI MboI/FastDigest 

MboI PsuI /FastDigest PsuI PsuI / 

FastDigest PsuI 

Eco130I /FastDigest StyI NheI/FastDigest NheI 

XbaI/FastDigest XbaI XmaJI /FastDigest AvrII 

 

Alw44I /FastDigest ApaLI HpyCH4V 

BamHI/FastDigest BamHI PsuI /FastDigest PsuI* 

 

BclI/FastDigest BclI Bsp143I /FastDigest Sau3AI 


PsuI /FastDigest PsuI 

Hin1I /FastDigest BsaHI Hin6I / 

FastDigest HinP1I SsiI /FastDigest 

AciI 

HpaII/FastDigest HpaII MspI /FastDigest MspI 


Bsp119I /FastDigest Bsp119I Bsu15I /FastDigest 

ClaI TaqI/FastDigest TaqI XmiI / 


BshTI /FastDigest AgeI Cfr10I /FastDigest 

BsrFI 

Cfr10I /FastDigest BsrFI MreI / 

FastDigest MreI 


sequence 

Bsp143I /FastDigest Sau3AI , DpnI/FastDigest DpnI, 

MboI/FastDigest MboI 

FspBI /FastDigest BfaI 





BglII/FastDigest BglII, Bsp143I /FastDigest Sau3AI , 

DpnI/FastDigest DpnI, MboI/FastDigest MboI, PsuI /FastDigest PsuI 

FauI, SsiI /FastDigest AciI 

BcnI /FastDigest NciI , Bme1390I /FastDigest ScrFI 

, BssKI, HpaII/FastDigest HpaII, MspI /FastDigest MspI 


BsaWI, BshTI /FastDigest AgeI , Cfr10I /FastDigest BsrFI 

, HpaII/FastDigest HpaII, MspI /FastDigest MspI 

Cfr10I /FastDigest BsrFI , HpaII/FastDigest HpaII, MspI 

/FastDigest MspI 


Cfr9I Eco88I /FastDigest AvaI 


Kpn2I /FastDigest Kpn2I BsaWI, HpaII/FastDigest HpaII, MspI /FastDigest MspI 

BshTI /FastDigest AgeI Cfr10I /FastDigest 

BsaWI, HpaII/FastDigest HpaII, MspI /FastDigest MspI 

BsrFI 







BsaWI, HpaII/FastDigest HpaII, Hpy188III, Kpn2I /FastDigest Kpn2I, 

Kpn2I /FastDigest Kpn2I 

MspI /FastDigest MspI 


ApaI/FastDigest ApaI Eco24I SduI 


Alw21I /FastDigest Alw21I SduI / 


Mph1103I /FastDigest NsiI HpyCH4V 


 

BsgI, HpyCH4V 


Bsh1285I /FastDigest 

PvuI/FastDigest PvuI SfaAI /FastDigest AsiSI 

BsiEI 

 

BshNI /FastDigest 

BanI 

BshNI /FastDigest 

BanI 

BseSI /FastDigest Bme1580I , SduI / 






Bsp1286I 






PacI/FastDigest PacI FastDigest MseI , Tru1I /FastDigest Tru1I 

SspDI 

Acc65I /FastDigest Acc65I 

Bsp1407I /FastDigest Bsp1407I Pfl23II / 

FastDigest BsiWI TatI/FastDigest TatI 


Bsh1285I /FastDigest BsiEI , Bsp143I /FastDigest 

Sau3AI , DpnI/FastDigest DpnI, MboI/FastDigest MboI, PvuI/ 






Acc65I /FastDigest Acc65I, BshNI /FastDigest BanI , 

BspLI /FastDigest NlaIV , Csp6I /FastDigest Csp6I, 

KpnI/FastDigest KpnI, RsaI/FastDigest RsaI 


 


192 


First 

Second 



sequence 


Cfr10I /FastDigest BsrFI BsaWI, BshTI /FastDigest AgeI , Cfr10I /FastDigest BsrFI 

BshTI /FastDigest 

AgeI 

 









Bsp119I /FastDigest 

Bsp119I 

Bsu15I /FastDigest ClaI 

TaqI/FastDigest TaqI XmiI /FastDigest AccI * 

 


Eco52I /FastDigest EagI BmgT120I, BsuRI /FastDigest HaeIII , Cfr13I / 

Bsp120I / 



BmgT120I, BsuRI /FastDigest HaeIII , Cfr13I / 

 

NotI/FastDigest NotI 

Acc65I /FastDigest Acc65I BshNI /FastDigest 

FastDigest Sau96I SatI /FastDigest Fnu4HI , 

SsiI /FastDigest AciI , TauI/FastDigest TauI 

BanI Pfl23II /FastDigest BsiWI Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI 

Bsp1407I /FastDigest 

Bsp1407I 


Bsp1407I /FastDigest Bsp1407I, Csp6I /FastDigest Csp6I, 

RsaI/FastDigest RsaI, TatI/FastDigest TatI 


BamHI/FastDigest BamHI BclI/FastDigest BclI 

Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI, TatI/FastDigest TatI 

Bsp143I /FastDigest BglII/FastDigest BglII MboI/FastDigest MboI Bsp143I /FastDigest Sau3AI , DpnI/FastDigest DpnI, 

Sau3AI PsuI /FastDigest PsuI PsuI /FastDigest PsuI* 

 


BspTI /FastDigest AflII 

SmoI 

 

BspTI /FastDigest AflII , FastDigest MseI , SmoI , 

Tru1I /FastDigest Tru1I 

Bsu15I /FastDigest 

ClaI 

Bsp119I /FastDigest Bsp119I 

TaqI/FastDigest TaqI XmiI /FastDigest AccI * 

 



PagI /FastDigest BspHI 

PscI 

CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII 

 

BseDI /FastDigest BsaJI , BtgI, CviAII, Eco130I /FastDigest 

Eco130I /FastDigest StyI NcoI/FastDigest NcoI 

StyI , FaiI, FatI, Hin1II /FastDigest NlaIII , NcoI/ 

 


MluI/FastDigest MluI Bsh1236I /FastDigest Bsh1236I, SsiI /FastDigest AciI 

 


PauI /FastDigest BssHII /FastDigest HinP1I , SsiI /FastDigest AciI 


BshTI /FastDigest AgeI BsaWI, BshTI /FastDigest AgeI , Cfr10I /FastDigest BsrFI 

Cfr10I /FastDigest 

BsrFI 

 





MreI /FastDigest MreI Cfr10I /FastDigest BsrFI , HpaII/FastDigest HpaII, MspI 

 


Kpn2I /FastDigest Kpn2I HpaII/FastDigest HpaII, MspI /FastDigest MspI 

BshTI /FastDigest AgeI Cfr10I /FastDigest BsrFI , HpaII/FastDigest HpaII, MspI 

Cfr10I /FastDigest 

BsrFI 

 





MreI /FastDigest MreI Cac8I, Cfr10I /FastDigest BsrFI , HpaII/FastDigest HpaII, MspI 

 

/FastDigest MspI, NgoMIV, PdiI /FastDigest NaeI 

Cfr42I Bsh1285I /FastDigest BsiEI 


AgeI Cfr10I /FastDigest BsrFI * 


Cfr10I /FastDigest BsrFI Kpn2I BcnI /FastDigest NciI , Bme1390I /FastDigest ScrFI 

/FastDigest Kpn2I MreI /FastDigest , BssKI, HpaII/FastDigest HpaII, MspI /FastDigest MspI 

Cfr9I 

MreI 

 


Eco88I /FastDigest AvaI 

, BmeT110I, BseDI /FastDigest BsaJI , BssKI, Cfr9I 

, Eco88I /FastDigest AvaI , HpaII/FastDigest HpaII, MspI 

/FastDigest MspI, SmaI/FastDigest SmaI 

 



First 

Second 



sequence 




 

Eco52I /FastDigest EagI 

Bsh1285I /FastDigest BsiEI , BsuRI /FastDigest 

HaeIII Eco52I /FastDigest EagI 



HaeIII Eco52I /FastDigest EagI , SatI 

/FastDigest Fnu4HI , SsiI /FastDigest AciI , TauI/ 





 

Eco52I /FastDigest EagI BsuRI /FastDigest HaeIII 


BsuRI /FastDigest HaeIII SatI /FastDigest 

Fnu4HI , SsiI /FastDigest AciI , TauI/FastDigest TauI 

Eco47I /FastDigest AvaII 

BmgT120I, Cfr13I /FastDigest Sau96I , Eco47I / 


CpoI /FastDigest RsrII FastDigest SanDI Psp5II /FastDigest PpuMI BmgT120I, BspLI /FastDigest NlaIV , Cfr13I /FastDigest 

 

Sau96I , Eco47I /FastDigest AvaII 

Psp5II /FastDigest PpuMI 


FastDigest AvaII , SetI 

Eco47I /FastDigest AvaII Psp5II / BmgT120I, Cfr13I /FastDigest Sau96I , Eco47I / 

CpoI /FastDigest RsrII FastDigest PpuMI 


FastDigest SanDI Psp5II /FastDigest PpuMI BmgT120I, BspLI /FastDigest NlaIV , Cfr13I /FastDigest 

 


Csp6I /FastDigest 

Csp6I 

NdeI/FastDigest NdeI FaiI 

XmiI /FastDigest AccI FaiI 

Eco130I /FastDigest 

StyI 


PscI 

NcoI/FastDigest NcoI 






StyI 

BcuI /FastDigest SpeI NheI/FastDigest NheI 

XbaI/FastDigest XbaI 

XmaJI /FastDigest AvrII 


BseDI /FastDigest BsaJI , Eco130I /FastDigest StyI 

, FspBI /FastDigest BfaI , XmaJI /FastDigest AvrII 

 

Eco24I SduI /FastDigest Bsp1286I Eco24I , SduI /FastDigest Bsp1286I 

Eco24I 

Alw21I /FastDigest Alw21I SacI/FastDigest SacI 







ApaI/FastDigest ApaI BseSI /FastDigest Bme1580I , Bsp120I /FastDigest Bsp120I, BspLI /FastDigest 

Eco24I SduI /FastDigest Bsp1286I * NlaIV , BsuRI /FastDigest HaeIII , Cfr13I / 

 


Bsp1286I 

Eco24I SduI /FastDigest Bsp1286I Eco24I , SduI /FastDigest Bsp1286I 

CpoI /FastDigest RsrII 



Eco47I /FastDigest FastDigest SanDI Psp5II /FastDigest PpuMI BmgT120I, BspLI /FastDigest NlaIV , Cfr13I /FastDigest 

AvaII 





CpoI /FastDigest RsrII Psp5II /FastDigest BmgT120I, Cfr13I /FastDigest Sau96I , Eco47I / 

Eco47I /FastDigest PpuMI 


AvaII FastDigest SanDI Psp5II /FastDigest PpuMI BmgT120I, BspLI /FastDigest NlaIV , Cfr13I /FastDigest 

 







EagI 

 


HaeIII Eco52I /FastDigest EagI 



HaeIII Eco52I /FastDigest EagI , SatI 

/FastDigest Fnu4HI , SsiI /FastDigest AciI , TauI/ 


 


194 


First 

Second 




AgeI Cfr10I /FastDigest BsrFI * 

sequence 

Cfr10I /FastDigest BsrFI Kpn2I BcnI /FastDigest NciI , Bme1390I /FastDigest ScrFI 

/FastDigest Kpn2I MreI /FastDigest , BssKI, HpaII/FastDigest HpaII, MspI /FastDigest MspI 

Eco88I /FastDigest MreI 

AvaI 


Cfr9I 

, BmeT110I, BseDI /FastDigest BsaJI , BssKI, Cfr9I 

, Eco88I /FastDigest AvaI , HpaII/FastDigest HpaII, MspI 

/FastDigest MspI, SmaI/FastDigest SmaI 

BmeT110I, Eco88I /FastDigest AvaI , SmoI , TaqI/ 

Eco88I /FastDigest SmoI XhoI/FastDigest XhoI FastDigest TaqI, XhoI/FastDigest XhoI 

AvaI SalI/FastDigest SalI TaqI/FastDigest TaqI 

SgrDI Hpy99I, TaqI/FastDigest TaqI 

EcoRI/FastDigest EcoRI 

 

MunI /FastDigest MfeI TasI /FastDigest 

Tsp509I 

XapI /FastDigest XapI 


EcoRI/FastDigest EcoRI, TasI /FastDigest Tsp509I , XapI 

/FastDigest XapI 

XapI /FastDigest XapI TasI /FastDigest Tsp509I , XapI /FastDigest XapI 

EcoRII FastDigest SexAI 

Bme1390I /FastDigest ScrFI , BssKI, EcoRII, MvaI / 

FastDigest MvaI, SetI 

EcoRII FastDigest SexAI 

Eco130I /FastDigest StyI * 



 

NcoI/FastDigest NcoI PagI /FastDigest 

BspHI PscI 


FspBI /FastDigest BfaI 

NdeI/FastDigest NdeI 

 

FaiI 

FastDigest HaeII * 

 

 

FastDigest HaeII , HhaI/FastDigest HhaI, Hin6I /FastDigest 

HinP1I 


FastDigest HaeII * 

 

 




Hin1I /FastDigest 

Hin6I /FastDigest HinP1I SsiI /FastDigest AciI 


 

BsaHI Psp1406I /FastDigest AclI MaeII, SetI, TaiI /FastDigest TaiI 

CseI /FastDigest HgaI , Hin1I /FastDigest BsaHI 



HhaI/FastDigest HhaI, Hin6I /FastDigest HinP1I 

 


BsaHI 

 




Hin1II /FastDigest 

NlaIII 

PaeI /FastDigest SphI XceI /FastDigest NspI 

XceI /FastDigest NspI 



HinP1I 

Hin1I /FastDigest BsaHI SsiI 



 



HpaII/FastDigest HpaII 

 



AciI 


MspI /FastDigest MspI SsiI /FastDigest AciI * 

 


 

BsaWI, HpaII/FastDigest HpaII, Hpy188III, Kpn2I /FastDigest Kpn2I, 

MspI /FastDigest MspI 

Kpn2I /FastDigest 

BshTI /FastDigest AgeI Cfr10I 

BsaWI, HpaII/FastDigest HpaII, MspI /FastDigest MspI 

/FastDigest BsrFI 

Kpn2I Cfr10I /FastDigest BsrFI MreI / 






 

Hin1I /FastDigest BsaHI Psp1406I / 



MauBI/FastDigest MauBI 

MluI/FastDigest MluI 

Bsh1236I /FastDigest Bsh1236I, HhaI/FastDigest HhaI, Hin6I 


 

PauI /FastDigest BssHII SgsI /FastDigest Bsh1236I /FastDigest Bsh1236I, Cac8I, HhaI/FastDigest HhaI, Hin6I 

AscI 

/FastDigest HinP1I , PauI /FastDigest BssHII 

 



First 

Second 



BamHI/FastDigest BamHI BclI/FastDigest BclI 

sequence 

MboI/FastDigest MboI BglII/FastDigest BglII Bsp143I /FastDigest Sau3AI Bsp143I /FastDigest Sau3AI , DpnI/FastDigest DpnI, 

 

PsuI /FastDigest PsuI PsuI / 

FastDigest PsuI 


AflIII, Bsh1236I /FastDigest Bsh1236I, MluI/FastDigest MluI 

MluI/FastDigest MluI Bsh1236I /FastDigest Bsh1236I 

 


PauI /FastDigest BssHII 

Alw21I /FastDigest Alw21I BseSI / 


Mph1103I /FastDigest 

NsiI 

FastDigest Bme1580I PstI/FastDigest PstI 

HpyCH4V 

SdaI /FastDigest SbfI SduI /FastDigest 

Bsp1286I 


BshTI /FastDigest AgeI Cfr10I Cfr10I /FastDigest BsrFI , HpaII/FastDigest HpaII, MspI 



Cfr10I /FastDigest BsrFI 

Cac8I, Cfr10I /FastDigest BsrFI , HpaII/FastDigest HpaII, MspI 


MreI /FastDigest 

MreI 




Cac8I, Cfr10I /FastDigest BsrFI , HpaII/FastDigest HpaII, 

 

MreI /FastDigest MreI, MspI /FastDigest MspI, NgoMIV, PdiI 

/FastDigest NaeI , SgrAI 

 


/FastDigest MspI, SgrAI 


 


Tru1I /FastDigest Tru1I VspI /FastDigest AseI 

 

FaiI 


 



MspI /FastDigest Hin1I /FastDigest BsaHI Hin6I / 

MspI 


AciI 


HpaII/FastDigest HpaII SsiI /FastDigest AciI HpaII/FastDigest HpaII, MspI /FastDigest MspI 

EcoRI/FastDigest EcoRI TasI /FastDigest Tsp509I 

MunI /FastDigest MfeI 

XapI /FastDigest XapI XapI / 

 

FastDigest XapI 



Hin1I /FastDigest BsaHI 



 


Hin1I /FastDigest BsaHI Hin1I /FastDigest BsaHI 



 

NcoI/FastDigest NcoI 

 


PscI 






NdeI/FastDigest NdeI 

 

Csp6I /FastDigest Csp6I FspBI /FastDigest BfaI 

FastDigest MseI Tru1I /FastDigest Tru1I FaiI 

VspI /FastDigest AseI 



/FastDigest BsrFI /FastDigest MspI 

Cfr10I /FastDigest BsrFI MreI / Cac8I, Cfr10I /FastDigest BsrFI , HpaII/FastDigest HpaII, MspI 






NheI/FastDigest NheI 

 

BcuI /FastDigest SpeI Eco130I /FastDigest 

StyI XbaI/FastDigest XbaI XmaJI / 





FastDigest Sau96I SatI /FastDigest Fnu4HI , 

TauI/FastDigest TauI 

NotI/FastDigest NotI 

 

 

BsuRI /FastDigest HaeIII SatI /FastDigest 

Fnu4HI , TauI/FastDigest TauI 


Eco52I /FastDigest EagI HaeIII Eco52I /FastDigest EagI , SatI 

/FastDigest Fnu4HI , TauI/FastDigest TauI 

 


196 


First 

Second 



sequence 

PacI/FastDigest PacI 

 

Bsh1285I /FastDigest BsiEI PvuI/FastDigest 

PvuI SfaAI /FastDigest AsiSI 


Hin1II /FastDigest NlaIII CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII 

PaeI /FastDigest SphI XceI /FastDigest NspI 

 

XceI /FastDigest NspI 

Cac8I, CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII , PaeI / 

FastDigest SphI , XceI /FastDigest NspI 

CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII , XceI / 


PagI /FastDigest 

BspHI 

Eco130I /FastDigest StyI * 

NcoI/FastDigest NcoI PscI 

 


PasI BseYI 

PauI /FastDigest 




BssHII MauBI/FastDigest MauBI SgsI /FastDigest AscI Bsh1236I /FastDigest Bsh1236I, Cac8I, HhaI/FastDigest HhaI, Hin6I 

 


Pfl23II /FastDigest 

BsiWI 


BanI Bsp1407I /FastDigest Bsp1407I Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI 

TatI/FastDigest TatI TatI/FastDigest TatI 

 

AflIII, CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII , PscI , XceI 

/FastDigest NspI 

PscI Eco130I /FastDigest StyI 

NcoI/FastDigest NcoI PagI /FastDigest BspHI 

 


Psp1406I /FastDigest 

AclI 

Hin1I /FastDigest BsaHI MaeII, SetI, TaiI /FastDigest TaiI 

CpoI /FastDigest RsrII Eco47I /FastDigest BmgT120I, Cfr13I /FastDigest Sau96I , Eco47I / 

Psp5II /FastDigest 

PpuMI 

AvaII 



BmgT120I, BspLI /FastDigest NlaIV , Cfr13I /FastDigest 

Sau96I , Eco47I /FastDigest AvaII , EcoO109I / 

FastDigest EcoO109I, Psp5II /FastDigest PpuMI 

CpoI /FastDigest RsrII Eco47I /FastDigest BmgT120I, Cfr13I /FastDigest Sau96I , Eco47I / 


PpuMI 

AvaII 





FastDigest EcoO109I, Psp5II /FastDigest PpuMI , SetI 


PpuMI 

CpoI /FastDigest RsrII Eco47I /FastDigest 

AvaII 


BmgT120I, BspLI /FastDigest NlaIV , Cfr13I / 

FastDigest Sau96I , Eco47I /FastDigest AvaII , FaqI 

/FastDigest BsmFI 



FastDigest EcoO109I, FaqI /FastDigest BsmFI , FastDigest 

SanDI , Psp5II /FastDigest PpuMI 

CpoI /FastDigest RsrII Eco47I /FastDigest BmgT120I, BspLI /FastDigest NlaIV , Cfr13I /FastDigest 

AvaII 




PpuMI 



FastDigest EcoO109I, FastDigest SanDI , Psp5II /FastDigest 

PpuMI 

 

BmeT110I, Eco88I /FastDigest AvaI , PspXI, SmoI , TaqI/ 

FastDigest TaqI, XhoI/FastDigest XhoI 

 

Eco88I /FastDigest AvaI SmoI BmeT110I, Eco88I /FastDigest AvaI , SmoI , TaqI/ 



SalI/FastDigest SalI TaqI/FastDigest TaqI 


 

AbsI, BmeT110I, Eco88I /FastDigest AvaI , MnlI/FastDigest 

MnlI, PspXI, SmoI , TaqI/FastDigest TaqI, XhoI/FastDigest XhoI 

 

Eco88I /FastDigest AvaI SmoI BmeT110I, Eco88I /FastDigest AvaI , MnlI/FastDigest MnlI, 


SmoI , TaqI/FastDigest TaqI, XhoI/FastDigest XhoI 

SalI/FastDigest SalI MnlI/FastDigest MnlI, TaqI/FastDigest TaqI 

SgrDI Hpy99I, MnlI/FastDigest MnlI, TaqI/FastDigest TaqI 

 

BmeT110I, Eco88I /FastDigest AvaI , PspXI, SmoI , TaqI/ 


 

Eco88I /FastDigest AvaI SmoI BmeT110I, Eco88I /FastDigest AvaI , SmoI , TaqI/ 







First 

Second 




sequence 

PstI/FastDigest PstI 

 

FastDigest Bme1580I Mph1103I /FastDigest 

NsiI SduI /FastDigest Bsp1286I * 

 

HpyCH4V 

SdaI /FastDigest SbfI BfmI /FastDigest SfcI , HpyCH4V, PstI/FastDigest PstI 

BamHI/FastDigest BamHI 



PsuI /FastDigest PsuI* BclI/FastDigest BclI Bsp143I /FastDigest Sau3AI Bsp143I /FastDigest Sau3AI , DpnI/FastDigest DpnI, 

 



BglII/FastDigest BglII 

BglII/FastDigest BglII, Bsp143I /FastDigest Sau3AI , 

DpnI/FastDigest DpnI, MboI/FastDigest MboI, PsuI /FastDigest PsuI 

BamHI/FastDigest BamHI, Bsp143I /FastDigest Sau3AI , 

BamHI/FastDigest BamHI 

BspLI /FastDigest NlaIV , BspPI , DpnI/FastDigest DpnI, 

PsuI /FastDigest PsuI* 

BclI/FastDigest BclI Bsp143I /FastDigest Sau3AI 

 





BglII/FastDigest BglII 


FastDigest DpnI, MboI/FastDigest MboI, PsuI /FastDigest PsuI 

PvuI/FastDigest PvuI 

 

Bsh1285I /FastDigest BsiEI SfaAI / 






SacI/FastDigest SacI 

 


Alw21I /FastDigest Alw21I Eco24I 




 



SmoI TaqI/FastDigest TaqI 

SalI/FastDigest SalI XhoI/FastDigest XhoI 

 

HincII /FastDigest HincII, Hpy8I /FastDigest Hpy8I, Hpy99I, 

SgrDI 

SalI/FastDigest SalI, TaqI/FastDigest TaqI, XmiI /FastDigest AccI 

 

CpoI /FastDigest RsrII Eco47I /FastDigest 

AvaII 

BmgT120I, BspLI /FastDigest NlaIV , Cfr13I / 

FastDigest Sau96I , Eco47I /FastDigest AvaII , FaqI 

/FastDigest BsmFI 


FastDigest SanDI * 

 



FastDigest EcoO109I, FaqI /FastDigest BsmFI , FastDigest 

SanDI , Psp5II /FastDigest PpuMI 




FastDigest EcoO109I, FaqI /FastDigest BsmFI , Psp5II / 

FastDigest PpuMI , SetI 

CpoI /FastDigest RsrII Eco47I /FastDigest BmgT120I, BspLI /FastDigest NlaIV , Cfr13I /FastDigest 

AvaII 



FastDigest SanDI * 

 



FastDigest EcoO109I, FastDigest SanDI , Psp5II /FastDigest 

PpuMI 





FastDigest EcoO109I, Psp5II /FastDigest PpuMI 

SdaI /FastDigest SbfI 

 

FastDigest Bme1580I Mph1103I /FastDigest 

NsiI SduI /FastDigest Bsp1286I * 

 

HpyCH4V 

PstI/FastDigest PstI BfmI /FastDigest SfcI , HpyCH4V, PstI/FastDigest PstI 

SduI /FastDigest 

Alw21I /FastDigest Alw21I 

Bsp1286I 


 


Eco24I 

Bsp1286I 

Eco24I , SduI /FastDigest Bsp1286I 


Bsp1286I 




SacI/FastDigest SacI 




BseSI /FastDigest Bme1580I 

Bsp1286I 



 


198 


First 

Second 



sequence 



SduI /FastDigest ApaI/FastDigest ApaI BseSI /FastDigest Bme1580I 


Bsp1286I Eco24I 


Bsp1286I 


Eco24I 

Bsp1286I 

Eco24I , SduI /FastDigest Bsp1286I 







Bsp1286I Mph1103I /FastDigest NsiI HpyCH4V 


 

BsgI, HpyCH4V 


BseSI /FastDigest Bme1580I 

Bsp1286I 




Alw21I /FastDigest Alw21I 

Bsp1286I 


 

AatII/FastDigest AatII TaiI /FastDigest TaiI 



 

SacI/FastDigest SacI SduI /FastDigest Bsp1286I 

 


FastDigest SexAI * 

 

EcoRII 



FastDigest SexAI * 

 

EcoRII 


FastDigest MvaI, SetI 

SfaAI /FastDigest 

AsiSI 

Bsh1285I /FastDigest BsiEI PvuI/FastDigest 

PvuI 






BshTI /FastDigest AgeI Cfr10I BsaWI, BshTI /FastDigest AgeI , Cfr10I /FastDigest BsrFI 



 







MreI /FastDigest MreI 


/FastDigest MspI, SgrAI 





 

Cac8I, Cfr10I /FastDigest BsrFI , HpaII/FastDigest HpaII, MspI 






Cac8I, Cfr10I /FastDigest BsrFI , HpaII/FastDigest HpaII, 

MreI /FastDigest MreI 


MreI /FastDigest MreI, MspI /FastDigest MspI, NgoMIV, PdiI 

/FastDigest NaeI , SgrAI 

SmoI Hpy99I, TaqI/FastDigest TaqI 

SgrDI 


HincII /FastDigest HincII, Hpy8I /FastDigest Hpy8I, Hpy99I, 

SalI/FastDigest SalI 

SalI/FastDigest SalI, TaqI/FastDigest TaqI, XmiI /FastDigest AccI 

 

SgsI /FastDigest AscI 




MauBI/FastDigest MauBI PauI /FastDigest BssHII Bsh1236I /FastDigest Bsh1236I, Cac8I, HhaI/FastDigest HhaI, Hin6I 

 


SmoI 


XhoI/ 

FastDigest XhoI 





SmoI BspTI /FastDigest AflII 

BspTI /FastDigest AflII , FastDigest MseI , SmoI , 

Tru1I /FastDigest Tru1I 

 



First 

Second 



sequence 

SsiI /FastDigest AciI 

 

 






HpaII/FastDigest HpaII MspI /FastDigest MspI HpaII/FastDigest HpaII, MspI /FastDigest MspI 

SsiI /FastDigest AciI 

 





SspDI BshNI /FastDigest BanI 




TaiI /FastDigest TaiI 

 

AatII/FastDigest AatII MaeII, SetI, TaiI /FastDigest TaiI 


 


Bsu15I /FastDigest ClaI XmiI /FastDigest 

AccI 


TasI /FastDigest 

Tsp509I 

EcoRI/FastDigest EcoRI MunI /FastDigest MfeI 

XapI /FastDigest XapI XapI /FastDigest TasI /FastDigest Tsp509I 

XapI 


TatI/FastDigest TatI* BanI Pfl23II /FastDigest BsiWI Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI 

 

 



Csp6I /FastDigest Csp6I, RsaI/FastDigest RsaI, TatI/FastDigest TatI 

TatI/FastDigest TatI* 

 

BanI Pfl23II /FastDigest BsiWI 

 



Bsp1407I /FastDigest Bsp1407I, Csp6I /FastDigest Csp6I, 

RsaI/FastDigest RsaI, TatI/FastDigest TatI 

Tru1I /FastDigest 

Tru1I 


FastDigest MseI VspI /FastDigest AseI 

 

FaiI 


VspI /FastDigest AseI NdeI/FastDigest NdeI FaiI 

 

FastDigest MseI Tru1I /FastDigest Tru1I FastDigest MseI , Tru1I /FastDigest Tru1I 

EcoRI/FastDigest EcoRI 

XapI /FastDigest XapI* 


 

Tsp509I 

TasI /FastDigest Tsp509I , XapI /FastDigest XapI 


EcoRI/FastDigest EcoRI 

XapI /FastDigest XapI* 

EcoRI/FastDigest EcoRI, TasI /FastDigest Tsp509I , XapI 

/FastDigest XapI 

 


Tsp509I 


XbaI/FastDigest XbaI 

 

BcuI /FastDigest SpeI Eco130I /FastDigest 

StyI NheI/FastDigest NheI XmaJI / 



Hin1II /FastDigest NlaIII 

XceI /FastDigest NspI 

 

PaeI /FastDigest SphI 


CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII , XceI / 


Hin1II /FastDigest NlaIII 

XceI /FastDigest NspI 

PaeI /FastDigest SphI 


Cac8I, CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII , PaeI / 

FastDigest SphI , XceI /FastDigest NspI 

XhoI/FastDigest XhoI 

 


SmoI 

 

SalI/FastDigest SalI 





XmaJI /FastDigest 

AvrII 

BcuI /FastDigest SpeI NheI/FastDigest NheI 

XbaI/FastDigest XbaI 



BseDI /FastDigest BsaJI , Eco130I /FastDigest StyI 

, FspBI /FastDigest BfaI , XmaJI /FastDigest AvrII 

 

XmiI /FastDigest AccI 

 

 

FaiI 


XmiI /FastDigest AccI 

Bsu15I /FastDigest ClaI TaqI/FastDigest TaqI 

 

 



200 

Recognition Sites Resulting from Fill-in of 5’-overhang and Self-ligation 

Note 


tion 

enzymes will cleave sequences in addition to the one listed. 

[ ] Enzymes that cleave the target both before and after the ligation. 



Table 1.23. Newly generated recognition sites resulting from fill-in of 5’-overhang and self-ligation. 

Restriction 

Recognition Newly generated Restriction enzymes that cleave 

enzyme 

sequence sequence after reaction the newly generated recognition sequence 

Bsh1285I /FastDigest BsiEI , Bsp143I /FastDigest Sau3AI , 

AbsI CCTCGAGG CCTCGATCGAGG 

DpnI/FastDigest DpnI, MboI/FastDigest MboI, [MnlI/FastDigest MnlI], PvuI/FastDigest PvuI, 

[TaqI/FastDigest TaqI] 

Acc65I /FastDigest Acc65I GGTACC GGTACGTACC 

[Csp6I /FastDigest Csp6I], Eco105I /FastDigest SnaBI , MaeII, Ppu21I 

/FastDigest BsaAI , [RsaI/FastDigest RsaI], SetI, TaiI /FastDigest TaiI 

AflIII* ACACGT ACACGCACGT [MaeII], [SetI], [TaiI /FastDigest TaiI] 

AflIII* ACATGT ACATGCATGT 

[CviAII], [FaiI], [FatI], [Hin1II /FastDigest NlaIII ], HpyCH4V, Mph1103I / 

FastDigest NsiI , [XceI /FastDigest NspI ] 

AflIII* ACGCGT ACGCGCGCGT 

[Bsh1236I /FastDigest Bsh1236I], Cac8I, HhaI/FastDigest HhaI, Hin6I / 

FastDigest HinP1I , MauBI/FastDigest MauBI, PauI /FastDigest BssHII 

AflIII* ACGTGT ACGTGCGTGT [MaeII], [SetI], [TaiI /FastDigest TaiI] 

Alw44I /FastDigest ApaLI 

 

GTGCAC GTGCATGCAC 

Cac8I, CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII , [HpyCH4V], PaeI /FastDigest 

SphI , XceI /FastDigest NspI 

BamHI/FastDigest BamHI GGATCC GGATCGATCC 

[Bsp143I /FastDigest Sau3AI ], [BspPI ], Bsu15I /FastDigest ClaI 

, [DpnI/FastDigest DpnI], [MboI/FastDigest MboI], TaqI/FastDigest TaqI 

BbvCI CCTCAGC CCTCATCAGC [MnlI/FastDigest MnlI] 

BclI/FastDigest BclI TGATCA TGATCGATCA 

[Bsp143I /FastDigest Sau3AI ], Bsu15I /FastDigest ClaI , [DpnI/ 

FastDigest DpnI], [MboI/FastDigest MboI], TaqI/FastDigest TaqI 

BcnI /FastDigest NciI CCSGG CCSSGG BseDI /FastDigest BsaJI 

[BcnI /FastDigest NciI ], [Bme1390I /FastDigest ScrFI ], BseDI 

BcnI /FastDigest NciI * CCCGG CCCCGG 

/FastDigest BsaJI , [BssKI], [HpaII/FastDigest HpaII], [MspI /FastDigest 

MspI] 

BcnI /FastDigest NciI * CCGGG CCGGGG 

[BcnI /FastDigest NciI ], [Bme1390I /FastDigest ScrFI ], BseDI / 

FastDigest BsaJI , [BssKI], [HpaII/FastDigest HpaII], [MspI /FastDigest MspI] 

BcuI /FastDigest SpeI ACTAGT ACTAGCTAGT AluI/FastDigest AluI FspBI /FastDigest BfaI ], SetI 

BfmI /FastDigest SfcI CTRYAG CTRYATRYAG FaiI 

BfmI /FastDigest SfcI * CTACAG CTACATACAG FaiI 

BfmI /FastDigest SfcI * CTATAG CTATATATAG [FaiI] 

BfmI /FastDigest SfcI * CTGCAG CTGCATGCAG 

Cac8I, CviAII, FaiI, FatI, Hin1II /FastDigest NlaIII , [HpyCH4V], PaeI /FastDigest 

SphI , XceI /FastDigest NspI 

BfmI /FastDigest SfcI * CTGTAG CTGTATGTAG FaiI 

BglII/FastDigest BglII AGATCT AGATCGATCT 



Bme1390I /FastDigest ScrFI 

 

CCNGG CCNNGG BseDI /FastDigest BsaJI 


* 

CCAGG CCAAGG BseDI /FastDigest BsaJI , Eco130I /FastDigest StyI 


* 

CCCGG CCCCGG 




* 

CCGGG CCGGGG 




* 

CCTGG CCTTGG BseDI /FastDigest BsaJI , Eco130I /FastDigest StyI 

BmeT110I CYCGRG CYCGCGRG Bsh1236I /FastDigest Bsh1236I 

BmgT120I GGNCC GGNNCC BspLI /FastDigest NlaIV 

BmgT120I* GGACC GGAACC BspLI /FastDigest NlaIV 

BmgT120I* GGCCC GGCCCC 

[BmgT120I], BspLI /FastDigest NlaIV , [BsuRI /FastDigest HaeIII ], 

[Cfr13I /FastDigest Sau96I 

BmgT120I* GGGCC GGGGCC 

[BmgT120I], BspLI /FastDigest NlaIV , [BsuRI /FastDigest HaeIII ], 

[Cfr13I /FastDigest Sau96I 

BmgT120I* GGTCC GGTTCC BspLI /FastDigest NlaIV 

Bpu10I/FastDigest Bpu10I* CCTCAGC CCTCATCAGC [MnlI/FastDigest MnlI] 

Bsh1285I /FastDigest BsiEI , BsuRI /FastDigest HaeIII , EaeI, 

BsaWI WCCGGW WCCGGCCGGW 

Eco52I /FastDigest EagI , [HpaII/FastDigest HpaII], [MspI /FastDigest 

MspI] 



R = G or A; K = G or T; B = C, G or T; 

Y = C or T; S = C or G; D = A, G or T; 

W = A or T; H = A, C or T; N = G, A, T or C. 

M = A or C; V = A, C or G;


Restriction 


enzyme 


BseYI CCCAGC CCCAGCCAGC 

BshNI /FastDigest BanI * GGCACC GGCACGCACC Cac8I 

BshNI /FastDigest BanI * GGCGCC GGCGCGCGCC 

Bsh1236I /FastDigest Bsh1236I, Cac8I, [HhaI/FastDigest HhaI], [Hin6I / 

FastDigest HinP1I ], PauI /FastDigest BssHII 

BshNI /FastDigest BanI * GGTACC GGTACGTACC 



BshNI /FastDigest BanI * GGTGCC GGTGCGTGCC Cac8I 

Bsh1285I /FastDigest BsiEI , BsuRI /FastDigest HaeIII , [Cfr10I 

BshTI /FastDigest AgeI ACCGGT ACCGGCCGGT 

/FastDigest BsrFI Eco52I /FastDigest EagI , [HpaII/ 

FastDigest HpaII], [MspI /FastDigest MspI] 

Bsp119I /FastDigest Bsp119I TTCGAA TTCGCGAA Bsh1236I /FastDigest Bsh1236I, Hpy188III, Bsp68I /FastDigest NruI 

[BmgT120I], [BsuRI /FastDigest HaeIII ], Cac8I, Cfr10I /FastDigest BsrFI 

Bsp120I /FastDigest Bsp120I GGGCCC GGGCCGGCCC 

, [Cfr13I /FastDigest Sau96I HpaII/FastDigest HpaII, MspI 


Bsp1407I /FastDigest Bsp1407I TGTACA TGTACGTACA 



Bsp143I /FastDigest Sau3AI 

 

GATC GATCGATC 



BspTI /FastDigest AflII CTTAAG CTTAATTAAG 

PacI/FastDigest PacI FastDigest MseI ], TasI /FastDigest 

Tsp509I , [Tru1I /FastDigest Tru1I] 

BssKI CCNGG CCNGGCCNGG 

[Bme1390I /FastDigest ScrFI ], [BssKI], BsuRI /FastDigest HaeIII 

 

BssKI* CCAGG CCAGGCCAGG 


EcoRII], [MvaI /FastDigest MvaI] 

[BcnI /FastDigest NciI ], [Bme1390I /FastDigest ScrFI ], BmgT120I, 

BssKI* CCCGG CCCGGCCCGG 

[BssKI], BsuRI /FastDigest HaeIII , Cfr13I /FastDigest Sau96I , 

HpaII/FastDigest HpaII], [MspI /FastDigest MspI] 


BssKI* CCGGG CCGGGCCGGG 

[BssKI], BsuRI /FastDigest HaeIII , Cfr13I /FastDigest Sau96I , 


BssKI* CCTGG CCTGGCCTGG 



Bsu15I /FastDigest ClaI ATCGAT ATCGCGAT Bsh1236I /FastDigest Bsh1236I, Hpy188III, Bsp68I /FastDigest NruI 

BtgI* CCATGG CCATGCATGG 




BtgI* CCGCGG CCGCGCGCGG 

FastDigest HinP1I , MauBI/FastDigest MauBI, [SsiI /FastDigest AciI ], PauI 

/FastDigest BssHII 


Cfr10I /FastDigest BsrFI RCCGGY RCCGGCCGGY 

/FastDigest BsrFI Eco52I /FastDigest EagI , [HpaII/ 


Cfr13I /FastDigest Sau96I 

* 

GGCCC GGCCGCCC 

[BsuRI /FastDigest HaeIII SatI /FastDigest Fnu4HI , SsiI 

/FastDigest AciI , TauI/FastDigest TauI 

Cfr13I /FastDigest Sau96I 

* 

GGGCC GGGCGGCC 

[BsuRI /FastDigest HaeIII SatI /FastDigest Fnu4HI , TauI/ 


[BcnI /FastDigest NciI ], [Bme1390I /FastDigest ScrFI ], Bsh1285I 

Cfr9I CCCGGG CCCGGCCGGG 

/FastDigest BsiEI , [BssKI], BsuRI /FastDigest HaeIII 


MspI] 

[BsuRI /FastDigest HaeIII ], Cac8I, Cfr10I /FastDigest BsrFI , 

EaeI YGGCCR YGGCCGGCCR 

HpaII/FastDigest HpaII, MspI /FastDigest MspI, NgoMIV, PdiI / 


CpoI /FastDigest RsrII * CGGACCG CGGACGACCG Bsh1285I /FastDigest BsiEI 

CpoI /FastDigest RsrII * CGGTCCG CGGTCGTCCG Bsh1285I /FastDigest BsiEI 

FastDigest SexAI ACCWGGT ACCWGGCCWGGT 



FastDigest SexAI * ACCAGGT ACCAGGCCAGGT 


EcoRII], [MvaI /FastDigest MvaI], [SetI] 

FastDigest SexAI * ACCTGGT ACCTGGCCTGGT 


EcoRII], [MvaI /FastDigest MvaI], [SetI] 

Csp6I /FastDigest Csp6I GTAC GTATAC 

Bst1107I /FastDigest BstZ17I , FaiI, Hpy8I /FastDigest Hpy8I, XmiI 

/FastDigest AccI 

CviAII CATG CATATG [FaiI], NdeI/FastDigest NdeI 

Eco130I /FastDigest StyI 

* 

CCATGG CCATGCATGG 



Eco130I /FastDigest StyI 

* 

CCTAGG CCTAGCTAGG AluI/FastDigest AluI FspBI /FastDigest BfaI ], SetI 

[Bsh1285I /FastDigest BsiEI ], [BsuRI /FastDigest HaeIII ], Cac8I, 

Eco52I /FastDigest EagI CGGCCG CGGCCGGCCG 

Cfr10I /FastDigest BsrFI Eco52I /FastDigest EagI ], 

FseI, HpaII/FastDigest HpaII, MspI /FastDigest MspI, NgoMIV, PdiI /FastDigest 

NaeI 

 


202 


Restriction 


enzyme 


Eco81I /FastDigest Bsu36I 

* 

CCTCAGG CCTCATCAGG [MnlI/FastDigest MnlI] 

Eco88I /FastDigest AvaI CYCGRG CYCGRYCGRG Bsh1285I /FastDigest BsiEI 

Eco88I /FastDigest AvaI * CCCGAG CCCGACCGAG Bsh1285I /FastDigest BsiEI 

[BcnI /FastDigest NciI ], [Bme1390I /FastDigest ScrFI ], Bsh1285I 

Eco88I /FastDigest AvaI * CCCGGG CCCGGCCGGG 

/FastDigest BsiEI , [BssKI], BsuRI /FastDigest HaeIII 


MspI] 

Eco88I /FastDigest AvaI * CTCGAG CTCGATCGAG 


DpnI/FastDigest DpnI, MboI/FastDigest MboI, PvuI/FastDigest PvuI, [TaqI/FastDigest TaqI] 

Eco88I /FastDigest AvaI * CTCGGG CTCGGTCGGG Bsh1285I /FastDigest BsiEI 

Eco91I /FastDigest Eco91I GGTNACC GGTNACGTNACC MaeII, [MaeIII], SetI, TaiI /FastDigest TaiI 

Eco91I /FastDigest Eco91I* GGTAACC GGTAACGTAACC MaeII, [MaeIII], SetI, TaiI /FastDigest TaiI 

Eco91I /FastDigest Eco91I* GGTCACC GGTCACGTCACC AjiI , MaeII, [MaeIII], [NmuCI /FastDigest NmuCI], SetI, TaiI /FastDigest TaiI 

Eco91I /FastDigest Eco91I* GGTGACC GGTGACGTGACC [HphI], MaeII, [MaeIII], [NmuCI /FastDigest NmuCI], SetI, TaiI /FastDigest TaiI 

Eco91I /FastDigest Eco91I* GGTTACC GGTTACGTTACC MaeII, [MaeIII], SetI, TaiI /FastDigest TaiI 

EcoO109I /FastDigest EcoO109I* RGGCCCY RGGCCGCCCY 

[BsuRI /FastDigest HaeIII SatI /FastDigest Fnu4HI , SsiI 

/FastDigest AciI , TauI/FastDigest TauI 

EcoO109I /FastDigest EcoO109I* RGGGCCY RGGGCGGCCY 

[BsuRI /FastDigest HaeIII SatI /FastDigest Fnu4HI , TauI/ 


EcoRI/FastDigest EcoRI GAATTC GAATTAATTC 

PdmI /FastDigest PdmI, FastDigest MseI , [TasI /FastDigest Tsp509I 

], Tru1I /FastDigest Tru1I, VspI /FastDigest AseI 

EcoRII CCWGG CCWGGCCWGG 



EcoRII* CCAGG CCAGGCCAGG 



EcoRII* CCTGG CCTGGCCTGG 



FatI CATG CATGCATG 



FspBI /FastDigest BfaI CTAG CTATAG BfmI /FastDigest SfcI , FaiI 

Hin1I /FastDigest BsaHI GRCGYC GRCGCGYC Bsh1236I /FastDigest Bsh1236I 

Hin6I /FastDigest HinP1I 

 

GCGC GCGCGC 



HindIII/FastDigest HindIII AAGCTT AAGCTAGCTT 

[AluI/FastDigest AluI], BspOI /FastDigest BmtI FspBI / 

FastDigest BfaI , NheI/FastDigest NheI, [SetI] 

HpaII/FastDigest HpaII CCGG CCGCGG 

BseDI /FastDigest BsaJI , Bsh1236I /FastDigest Bsh1236I, BtgI, Cfr42I 

, MspA1I, SsiI /FastDigest AciI 

Bsh1285I /FastDigest BsiEI , BsuRI /FastDigest HaeIII , EaeI, 

Kpn2I /FastDigest Kpn2I TCCGGA TCCGGCCGGA 


MspI] 

MaeII ACGT ACGCGT AflIII, Bsh1236I /FastDigest Bsh1236I, MluI/FastDigest MluI 

MaeIII GTNAC GTNACGTNAC MaeII, [MaeIII], SetI, TaiI /FastDigest TaiI 

MaeIII* GTAAC GTAACGTAAC MaeII, [MaeIII], SetI, TaiI /FastDigest TaiI 

MaeIII* GTCAC GTCACGTCAC AjiI , MaeII, [MaeIII], [NmuCI /FastDigest NmuCI], SetI, TaiI /FastDigest TaiI 

MaeIII* GTGAC GTGACGTGAC MaeII, [MaeIII], [NmuCI /FastDigest NmuCI], SetI, TaiI /FastDigest TaiI 

MaeIII* GTTAC GTTACGTTAC MaeII, [MaeIII], SetI, TaiI /FastDigest TaiI 

MauBI/FastDigest MauBI CGCGCGCG CGCGCGCGCGCG 

[Bsh1236I /FastDigest Bsh1236I], [Cac8I], [HhaI/FastDigest HhaI], [Hin6I / 

FastDigest HinP1I ], [MauBI/FastDigest MauBI], [PauI /FastDigest BssHII ] 

MboI/FastDigest MboI GATC GATCGATC 



MluI/FastDigest MluI ACGCGT ACGCGCGCGT 


FastDigest HinP1I , MauBI/FastDigest MauBI, PauI /FastDigest BssHII 

Bsh1285I /FastDigest BsiEI , BsuRI /FastDigest HaeIII , [Cac8I], 

MreI /FastDigest MreI CGCCGGCG CGCCGGCCGGCG 

[Cfr10I /FastDigest BsrFI Eco52I /FastDigest EagI , 

[HpaII/FastDigest HpaII], [MspI /FastDigest MspI], [NgoMIV], [PdiI /FastDigest 

NaeI ] 

FastDigest MseI TTAA TTATAA AanI /FastDigest PsiI , FaiI 

MspI /FastDigest MspI CCGG CCGCGG 

BseDI /FastDigest BsaJI , Bsh1236I /FastDigest Bsh1236I, BtgI, Cfr42I 

, MspA1I, SsiI /FastDigest AciI 

MunI /FastDigest MfeI CAATTG CAATTAATTG 

FastDigest MseI , [TasI /FastDigest Tsp509I ], Tru1I /FastDigest 

Tru1I, VspI /FastDigest AseI 

MvaI /FastDigest MvaI CCWGG CCWWGG BseDI /FastDigest BsaJI , Eco130I /FastDigest StyI 

MvaI /FastDigest MvaI* CCAGG CCAAGG BseDI /FastDigest BsaJI , Eco130I /FastDigest StyI 

MvaI /FastDigest MvaI* CCTGG CCTTGG BseDI /FastDigest BsaJI , Eco130I /FastDigest StyI 

NarI GGCGCC GGCGCGCC 

Bsh1236I /FastDigest Bsh1236I, Cac8I, [HhaI/FastDigest HhaI], [Hin6I /FastDigest 

HinP1I ], SgsI /FastDigest AscI , PauI /FastDigest BssHII 

NcoI/FastDigest NcoI CCATGG CCATGCATGG 





Restriction 


enzyme 


NdeI/FastDigest NdeI CATATG CATATATG [FaiI] 

Bsh1285I /FastDigest BsiEI , BsuRI /FastDigest HaeIII , [Cac8I], 

NgoMIV GCCGGC GCCGGCCGGC 

[Cfr10I /FastDigest BsrFI Eco52I /FastDigest EagI , 

[HpaII/FastDigest HpaII], [MspI /FastDigest MspI], [NgoMIV], [PdiI /FastDigest 

NaeI ] 

NheI/FastDigest NheI GCTAGC GCTAGCTAGC 

AluI/FastDigest AluI, [BspOI /FastDigest BmtI FspBI / 

FastDigest BfaI ], [NheI/FastDigest NheI], SetI 

NmuCI /FastDigest NmuCI GTSAC GTSACGTSAC MaeII, [MaeIII], [NmuCI /FastDigest NmuCI], SetI, TaiI /FastDigest TaiI 

NmuCI /FastDigest NmuCI* GTCAC GTCACGTCAC AjiI , MaeII, [MaeIII], [NmuCI /FastDigest NmuCI], SetI, TaiI /FastDigest TaiI 

NmuCI /FastDigest NmuCI* GTGAC GTGACGTGAC MaeII, [MaeIII], [NmuCI /FastDigest NmuCI], SetI, TaiI /FastDigest TaiI 

[Bsh1285I /FastDigest BsiEI ], [BsuRI /FastDigest HaeIII ], Cac8I, 

Cfr10I /FastDigest BsrFI Eco52I /FastDigest EagI ], 

NotI/FastDigest NotI GCGGCCGC GCGGCCGGCCGC FseI, HpaII/FastDigest HpaII, MspI /FastDigest MspI, NgoMIV, PdiI /FastDigest 

NaeI , [SatI /FastDigest Fnu4HI ], [SsiI /FastDigest AciI ], [TauI/ 

FastDigest TauI] 

PagI /FastDigest BspHI TCATGA TCATGCATGA 



PasI* CCCAGGG CCCAGCAGGG BseYI, EcoP15I 

PauI /FastDigest BssHII GCGCGC GCGCGCGCGC 


FastDigest HinP1I ], MauBI/FastDigest MauBI, [PauI /FastDigest BssHII ] 

Pfl23II /FastDigest BsiWI 

 

CGTACG CGTACGTACG 

[Csp6I /FastDigest Csp6I], Eco105I /FastDigest SnaBI , MaeII, [Pfl23II 

/FastDigest BsiWI ], Ppu21I /FastDigest BsaAI , [RsaI/FastDigest 

RsaI], SetI, TaiI /FastDigest TaiI 

PfoI/FastDigest PfoI TCCNGGA TCCNGGCCNGGA 


 

PfoI/FastDigest PfoI* TCCAGGA TCCAGGCCAGGA 




PfoI/FastDigest PfoI* TCCCGGA TCCCGGCCCGGA [BssKI], BsuRI /FastDigest HaeIII , Cfr13I /FastDigest Sau96I , 



PfoI/FastDigest PfoI* TCCGGGA TCCGGGCCGGGA [BssKI], BsuRI /FastDigest HaeIII , Cfr13I /FastDigest Sau96I , 


PfoI/FastDigest PfoI* TCCTGGA TCCTGGCCTGGA 



PscI ACATGT ACATGCATGT 


FastDigest NsiI , [XceI /FastDigest NspI ] 

Psp1406I /FastDigest AclI 

 

AACGTT AACGCGTT AflIII, Bsh1236I /FastDigest Bsh1236I, MluI/FastDigest MluI 

PspXI VCTCGAGB VCTCGATCGAGB 



PsuI /FastDigest PsuI RGATCY RGATCGATCY 



PsyI /FastDigest PsyI GACNNNGTC GACNNNNGTC BoxI /FastDigest PshAI 

PsyI /FastDigest PsyI* GACNANGTC GACNAANGTC BoxI /FastDigest PshAI 

PsyI /FastDigest PsyI* GACNCNGTC GACNCCNGTC BoxI /FastDigest PshAI 

PsyI /FastDigest PsyI* GACNGNGTC GACNGGNGTC BoxI /FastDigest PshAI 

PsyI /FastDigest PsyI* GACNTNGTC GACNTTNGTC BoxI /FastDigest PshAI 

SalI/FastDigest SalI GTCGAC GTCGATCGAC 



FastDigest SanDI * GGGACCC GGGACGACCC [FaqI /FastDigest BsmFI ] 

SatI /FastDigest Fnu4HI GCNGC GCNNGC Cac8I 

SatI /FastDigest Fnu4HI * GCAGC GCAAGC Cac8I 

SatI /FastDigest Fnu4HI * GCCGC GCCCGC Cac8I, FauI, [SsiI /FastDigest AciI ] 

SatI /FastDigest Fnu4HI * GCGGC GCGGGC Cac8I 

SatI /FastDigest Fnu4HI * GCTGC GCTTGC Cac8I 


SgrAI CRCCGGYG CRCCGGCCGGYG /FastDigest BsrFI Eco52I /FastDigest EagI , [HpaII/ 



SgrDI CGTCGACG CGTCGATCGACG 

DpnI/FastDigest DpnI, [Hpy99I], MboI/FastDigest MboI, PvuI/FastDigest PvuI, [TaqI/ 

FastDigest TaqI] 

SgsI /FastDigest AscI GGCGCGCC GGCGCGCGCGCC 


FastDigest HinP1I ], MauBI/FastDigest MauBI, [PauI /FastDigest BssHII ] 

SmoI * CTCGAG CTCGATCGAG 



SmoI * CTTAAG CTTAATTAAG 

PacI/FastDigest PacI FastDigest MseI ], TasI /FastDigest 

Tsp509I , [Tru1I /FastDigest Tru1I] 

SsiI /FastDigest AciI CCGC CCGCGC 

Bsh1236I /FastDigest Bsh1236I, HhaI/FastDigest HhaI, Hin6I /FastDigest 

HinP1I , [SsiI /FastDigest AciI ] 

 


204 


Restriction 


enzyme 


SspDI GGCGCC GGCGCGCGCC 



TaqI/FastDigest TaqI TCGA TCGCGA Bsh1236I /FastDigest Bsh1236I, Hpy188III, Bsp68I /FastDigest NruI 

TasI /FastDigest Tsp509I 

 

AATT AATTAATT 



TatI/FastDigest TatI WGTACW WGTACGTACW 



Tru1I /FastDigest Tru1I TTAA TTATAA AanI /FastDigest PsiI , FaiI 

TseI GCWGC GCWGCWGC [SatI /FastDigest Fnu4HI ], [TseI] 

TseI* GCAGC GCAGCAGC 

[BseXI /FastDigest BseXI], EcoP15I, [Lsp1109I /FastDigest BbvI ], [SatI 

/FastDigest Fnu4HI ], [TseI] 

TseI* GCTGC GCTGCTGC [SatI /FastDigest Fnu4HI ], [TseI] 

VspI /FastDigest AseI ATTAAT ATTATAAT AanI /FastDigest PsiI , FaiI 

XapI /FastDigest XapI RAATTY RAATTAATTY 



XbaI/FastDigest XbaI TCTAGA TCTAGCTAGA AluI/FastDigest AluI FspBI /FastDigest BfaI ], SetI 

XhoI/FastDigest XhoI CTCGAG CTCGATCGAG 



XmaJI /FastDigest AvrII CCTAGG CCTAGCTAGG AluI/FastDigest AluI FspBI /FastDigest BfaI ], SetI 

XmiI /FastDigest AccI * GTATAC GTATATAC [FaiI] 

XmiI /FastDigest AccI * GTCGAC GTCGCGAC Bsh1236I /FastDigest Bsh1236I, Hpy188III, Bsp68I /FastDigest NruI 

XmiI /FastDigest AccI * GTCTAC GTCTCTAC Alw26I /FastDigest Alw26I 

Recognition Sites Resulting from Removal of 3’-overhang and Self-ligation 

Table 1.24. Newly generated recognition sites resulting from removal of 3’-overhang and self-ligation. 

Restriction 


enzyme 

sequence 

sequence after reaction the newly generated recognition sequence 

FastDigest DrdI /DrdI GACNNNNNNGTC GACNNNNGTC BoxI /FastDigest PshAI 

AdeI /FastDigest DraIII 

 

CACNNNGTG CACGTG 

Eco72I /FastDigest PmlI , MaeII, Ppu21I /FastDigest BsaAI , SetI, 

TaiI /FastDigest TaiI 

AgsI TTSAA TTAA FastDigest MseI , Tru1I /FastDigest Tru1I 

BglI/FastDigest BglI GCCNNNNNGGC GCCNNGGC BseDI /FastDigest BsaJI 

Bsh1285I /FastDigest 

BsiEI 

CGRYCG CGCG Bsh1236I /FastDigest Bsh1236I 

CaiI /FastDigest AlwNI 

 

CAGNNNCTG CAGCTG AluI/FastDigest AluI PvuII/FastDigest PvuII, SetI 

Cfr42I CCGCGG CCGG HpaII/FastDigest HpaII, MspI /FastDigest MspI 

Eam1105I /FastDigest 

Eam1105I 

GACNNNNNGTC GACNNNNGTC BoxI /FastDigest PshAI 

FseI GGCCGGCC GGCC [BsuRI /FastDigest HaeIII 

Hpy188I TCNGA TCGA TaqI/FastDigest TaqI 

PacI/FastDigest PacI 

 

TTAATTAA TTATAA AanI /FastDigest PsiI , FaiI 

PvuI/FastDigest PvuI CGATCG CGCG Bsh1236I /FastDigest Bsh1236I 

SdaI /FastDigest SbfI CCTGCAGG CCGG HpaII/FastDigest HpaII, MspI /FastDigest MspI 

SfaAI /FastDigest AsiSI 

 

GCGATCGC GCGCGC 

Bsh1236I /FastDigest Bsh1236I, Cac8I, HhaI/FastDigest HhaI, Hin6I / 

FastDigest HinP1I , PauI /FastDigest BssHII 

SfiI/FastDigest SfiI GGCCNNNNNGGCC GGCCNNGGCC BseDI /FastDigest BsaJI , [BsuRI /FastDigest HaeIII 

TaaI /FastDigest TaaI ACNGT ACGT MaeII, SetI, TaiI /FastDigest TaiI 

Note 





R = G or A; K = G or T; B = C, G or T; M = A or C 

Y = C or T; S = C or G; D = A, G or T; V = A, C or G; 

W = A or T; H = A, C or T; N = G, A, T or C.

Selection Guides 

Alphabetic List of Commercially Available Restriction Enzymes 

Note 

[ ] 

m Isoschizomers with different sensitivity to methylation. 

* Thermo Scientific restriction enzymes are shown in bold. 

FastDigest enzymes are shown in purple. 

Conventional enzymes are shown in orange. 

Table 1.25. Commercially available restriction enzymes. 







S = C or G; 

Enzyme Specificity 5’3’ FastDigest enzyme Page Conventional enzyme Page 

Commercially available 

isoschizomers from other vendors 

AanI TTATAA FastDigest PsiI 57 AanI 82 PsiI 

AarI AarI 82 

AasI GACNNNNNNGTC FastDigest DrdI 36 DrdI, DseDI 

AatI AGGCCT FastDigest StuI 66 Eco147I 114 PceI, SseBI, StuI 

AatII GACGTC FastDigest AatII 13 AatII 82 [ZraIm ] 

AbsI CCTCGAGG 

Acc16I TGCGCA FastDigest FspI 41 NsbI 132 AviII, FspI 

Acc36I FastDigest BspMI 31 BveI 104 BfuAI, BspMI 

Acc65I GGTACC 

FastDigest Acc65I, 

[FastDigest KpnIm 13 

45 

Acc65I , 

[KpnIm 83 

123 

Asp718Im AccB1I GGYRCC FastDigest BanI 21 BshNI 97 BanI, BspT107I 

AccB7I CCANNNNNTGG FastDigest PflMI 56 Van91I 152 BasI, PflMI 

AccBSI FastDigest BsrBI 32 MbiI 125 BsrBI 

AccI GTMKAC FastDigest AccI 13 XmiI 154 AccI, FblI 

AccII CGCG FastDigest Bsh1236I 27 Bsh1236I 97 BspFNI, BstFNI, BstUI, MvnI 

AccIII TCCGGA FastDigest Kpn2Im 46 Kpn2I m 123 Aor13HI, BseAIm , Bsp13I, BspEIm , MroIm AciI FastDigest AciI 14 SsiI 147 AciI, BspACI 

AclI AACGTT FastDigest AclI 14 Psp1406I 138 AclI 

AclWI BspPI 101 AlwI 

AcoI YGGCCR EaeI 

AcsI RAATTY FastDigest XapI 70 XapI 153 ApoI 

AcuI FastDigest AcuI 14 Eco57I 112 AcuI 

AcvI CACGTG FastDigest PmlI 56 Eco72I 112 BbrPI, PmaCI, PmlI, PspCI 

AcyI GRCGYC FastDigest BsaHI 26 Hin1I 119 BsaHI, BssNI, BstACI, Hsp92I 

AdeI CACNNNGTG FastDigest DraIII 36 AdeI 83 DraIII 

AfaI GTAC 

[FastDigest Csp6Im FastDigest RsaI 

34 

60 

[Csp6I m RsaI 

107 

140 

m ], [RsaNI] 

AfeI AGCGCT FastDigest AfeI 15 Eco47III 111 AfeI, Aor51HI 

AfiI CCNNNNNNNGG FastDigest BslI 28 BseLI 95 Bsc4I, BslI 

AflII CTTAAG FastDigest AflII 15 BspTI 101 AflII, BfrI, Bst98I, BstAFI, MspCI, Vha464I 

AflIII ACRYGT 

AgeI ACCGGT FastDigest AgeI 15 BshTI 98 AgeI, AsiGI, CspAIm , PinAI 

AgsI TTSAA 

AhdI GACNNNNNGTC FastDigest Eam1105I 37 Eam1105I 108 AspEI, BmeRI, DriI, EclHKI 

AhlI ACTAGT FastDigest SpeI 65 BcuI 89 SpeI 

AjiI AjiI 84 BmgBI, BtrI 

AjnI CCWGG [FastDigest MvaI 50 

EcoRIIm , 

[MvaI 

116 

129 

[BseBI], [Bst2UI], [BstNI], [BstOI], 

Psp6I, PspGIm AjuI 7 FastDigest AjuI 16 AjuI 84 

AleI CACNNNNGTG FastDigest AleI 16 OliI 133 AleI 

AlfI 6 AlfI 85 

AloI 6 AloI 85 

AluI AGCT FastDigest AluI 16 AluI 85 AluBI 

AluBI AGCT FastDigest AluI 16 AluI 85 

Alw21I GWGCWC FastDigest Alw21I 17 Alw21I 86 Bbv12I, BsiHKAI 

Alw26I FastDigest Alw26I 17 Alw26I 86 BsmAIm , BstMAI 

Alw44I GTGCAC FastDigest ApaLI 18 Alw44I 86 ApaLIm , VneI 

AlwI BspPI 101 AclWI 

AlwNI CAGNNNCTG FastDigest AlwNI 17 CaiI 104 AlwNI 

Ama87I CYCGRG FastDigest AvaI 19 Eco88I 113 AvaI, [BmeT110I], BsiHKCI, BsoBI 

Aor13HI TCCGGA FastDigest Kpn2I 46 Kpn2I 123 AccIII, BseAI, Bsp13I, BspEI, MroI 

Aor51HI AGCGCT FastDigest AfeI 15 Eco47III 111 AfeI 

ApaI GGGCCC 

FastDigest ApaI, 

[FastDigest Bsp120I 

18 

30 

ApaI, 

[Bsp120I 

87 

99 

[PspOMI] 

 


206 





ApaLI GTGCAC FastDigest ApaLI m 18 Alw44I m 86 ApaLI, VneI 

ApeKI GCWGC TseI 

ApoI RAATTY FastDigest XapI 70 XapI 153 AcsI 

ArsI 6 AscI GGCGCGCC FastDigest AscI 18 SgsI 145 AscI, PalAI 

AseI ATTAAT FastDigest AseI 19 VspI 152 AseI, PshBI 

AsiGI ACCGGT FastDigest AgeI 15 BshTI 98 AgeI, CspAI, PinAI 

AsiSI GCGATCGC FastDigest AsiSI 19 SfaAI 144 AsiSI, RgaI, SgfI 

Asp700I GAANNNNTTC FastDigest PdmI 55 PdmI 135 MroXI, XmnI 

Asp718I GGTACC 

FastDigest Acc65Im , 

[FastDigest KpnIm 13 

45 

Acc65I m , 

[KpnIm 83 

123 

AspA2I CCTAGG FastDigest AvrII 20 XmaJI 154 AvrII, BlnI 

AspEI GACNNNNNGTC FastDigest Eam1105I 37 Eam1105I 108 AhdI, BmeRI, DriI, EclHKI 

AspI GACNNNGTC FastDigest PsyI 59 PsyI 139 PflFI, Tth111I 

AspLEI GCGC 

FastDigest HhaI, 

[FastDigest HinP1I 

42 

44 

HhaI, 

[Hin6I 

119 

120 

BstHHI, CfoI, [HinP1I], [HspAI] 

AspS9I GGNCC FastDigest Sau96I 62 Cfr13I 105 [BmgT120I], PspPI, Sau96I 

AssI AGTACT FastDigest ScaI 63 ScaI 142 BmcAI, ZrmI 

AsuC2I CCSGG FastDigest NciI 51 BcnI 89 BpuMI, NciI 

AsuHPI HphI 122 

AsuII TTCGAA FastDigest Bsp119Im 29 Bsp119I m 99 

Bpu14I, BspT104Im , BstBIm , Csp45I, 

NspVm , SfuI 

AsuNHI GCTAGC 


[FastDigest BmtI 


24 

52 

[BspOI 

NheI 

AvaI CYCGRG FastDigest AvaI m 19 Eco88I m 113 

Ama87I, AvaI, [BmeT110Im ], BsiHKCI, 

BsoBIm AvaII GGWCC FastDigest AvaII 20 Eco47I 110 AvaII, Bme18I, SinI, VpaK11BI 

AviII TGCGCA FastDigest FspI 41 NsbI 132 Acc16I, FspI 

AvrII CCTAGG FastDigest AvrII 20 XmaJI 154 AspA2I, AvrII, BlnI 

AxyI CCTNAGG FastDigest Bsu36I 34 Eco81I 113 Bse21I, Bsu36I 

BaeGI GKGCMC FastDigest Bme1580I 23 BseSI 96 BstSLI 

BaeI 4 BalI TGGCCA FastDigest MscI 49 MlsI 127 MluNI, MscI, Msp20I 

BamHI GGATCC FastDigest BamHI 20 BamHI 88 

BanI GGYRCC FastDigest BanI 21 BshNI 97 AccB1I, BanI, BspT107I 

BanII GRGCYC Eco24I 109 EcoT38I, FriOI 

BanIII ATCGAT FastDigest ClaI 34 Bsu15I 103 

Bsa29I, BseCI, BshVI, BspDI, BspXI, 

BsuTUI, ClaI 

BarI 6 BasI CCANNNNNTGG FastDigest PflMI 56 Van91I 152 AccB7I, PflMI 

BauI BauI 88 BssSI, Bst2BI 

BbeI GGCGCC [FastDigest EheI 40 

[EheI 

[SspDI 

116 

148 

[DinI], [EgeI], [KasI], [Mly113I], [NarI], 

[SfoIm ] 

BbrPI CACGTG FastDigest PmlI 56 Eco72I 112 AcvI, PmaCI, PmlI, PspCI 

BbsI FastDigest BbsI 21 BpiI 92 BbsI, BpuAI, BstV2I 

BbuI GCATGC FastDigest SphI 66 PaeI 133 SphI 

Bbv12I GWGCWC FastDigest Alw21I 17 Alw21I 86 BsiHKAI 

BbvCI 

BbvI 

FastDigest BbvI , 


21 

27 

Lsp1109I , 

BseXI 

100 

131 

124 

96 

[BmtI] 

BbvI, BstV1I 

BccI 

BceAI 

BcgI 6 BciVI BfuI 90 

BclI TGATCA FastDigest BclI 22 BclI 89 FbaI, Ksp22I 

BcnI CCSGG FastDigest NciI 51 BcnI 89 AsuC2I, BpuMI, NciI 

BcuI ACTAGT FastDigest SpeI 65 BcuI 89 AhlI, SpeI 

BdaI 6 BfaI CTAG FastDigest BfaI 22 FspBI 118 BfaI, MaeI, XspI 

BfiI BmrI, BmuI 

BfmI CTRYAG FastDigest SfcI 64 BfmI 90 BstSFI, SfcI 

BfrI CTTAAG FastDigest AflII 15 BspTI 101 AflII, Bst98I, BstAFI, MspCI, Vha464I 

BfuAI FastDigest BspMI 31 BveI 104 Acc36I, BspMI 

BfuCI GATC 

FastDigest Sau3AI , 

FastDigest MboIm 62 

46 

Bsp143I , 

MboIm 99 

126 

BssMI, [BstKTIm ], BstMBI, DpnIIm , 

Kzo9I, NdeIIm , Sau3AIm BfuI BfuI 90 BciVI 

BglI GCCNNNNNGGC FastDigest BglI 22 BglI 90





BglII AGATCT FastDigest BglII 23 BglII 91 

BisI GCNGC [BlsI], GluI 

BlnI CCTAGG FastDigest AvrII 20 XmaJI 154 AspA2I, AvrII 

BlpI GCTNAGC FastDigest BlpI 23 Bpu1102I 93 BlpI, Bsp1720I, CelII 

BlsI GCNGC [BisI], [GluI] 

BmcAI AGTACT FastDigest ScaI 63 ScaI 142 AssI, ZrmI 

Bme1390I CCNGG FastDigest ScrFI 63 Bme1390I 91 

BmrFI, [BssKI], [BstSCI], MspR9I, ScrFI, 

[StyD4I] 

Bme1580I GKGCMC FastDigest Bme1580I 23 BseSI 96 BaeGI, BstSLI 

Bme18I GGWCC FastDigest AvaII 20 Eco47I 110 AvaII, SinI, VpaK11BI 

BmeRI GACNNNNNGTC FastDigest Eam1105I 37 Eam1105I 108 AhdI, AspEI, DriI, EclHKI 

BmeT110I CYCGRG [FastDigest AvaI 

 

m 19 [Eco88I m 113 [Ama87I], [AvaIm ], [BsiHKCI], [BsoBI] 

BmgBI AjiI 84 BtrI 

BmgT120I GGNCC [FastDigest Sau96I 62 [Cfr13I 105 [AspS9I], [PspPI], [Sau96I] 

BmiI GGNNCC FastDigest NlaIV 53 BspLI 100 NlaIV, PspN4I 

BmrFI CCNGG FastDigest ScrFI 63 Bme1390I 91 [BssKI], [BstSCI], MspR9I, ScrFI, [StyD4I] 

BmrI BmuI 

BmtI GCTAGC 

FastDigest BmtI , 

[FastDigest NheIm 24 

52 

BspOI , 

[NheIm 100 

131 

[AsuNHI], BmtI 

BmuI BmrI 

BoxI GACNNNNGTC FastDigest PshAI 57 BoxI 92 BstPAI, PshAI 

BpiI FastDigest BbsI 21 BpiI 92 BbsI, BpuAI, BstV2I 

BplI 5 FastDigest BplI 24 BplI 92 

BpmI FastDigest BpmI m 24 GsuI m 118 BpmI 

Bpu10I FastDigest Bpu10I 25 Bpu10I 93 

Bpu1102I GCTNAGC FastDigest BlpI 23 Bpu1102I 93 BlpI, Bsp1720I, CelII 

Bpu14I TTCGAA FastDigest Bsp119I 29 Bsp119I 99 AsuII, BspT104I, BstBI, Csp45I, NspV, SfuI 

BpuAI FastDigest BbsI 21 BpiI 92 BbsI, BstV2I 

BpuEI 

BpuMI CCSGG FastDigest NciI 51 BcnI 89 AsuC2I, NciI 

BpvUI CGATCG FastDigest PvuI 59 PvuI 139 MvrI, Ple19I 

Bsa29I ATCGAT FastDigest ClaI 34 Bsu15I 103 

BanIII, BseCI, BshVI, BspDI, BspXI, 

BsuTUI, ClaI 

BsaAI YACGTR FastDigest BsaAI 25 Ppu21I 137 BsaAI, BstBAI 

BsaBI GATNNNNATC FastDigest BsaBI 25 BseJI 94 BsaBI, Bse8I 

BsaHI GRCGYC FastDigest BsaHI m 26 Hin1I m 119 AcyI, BsaHI, BssNI, BstACI, Hsp92I 

BsaI FastDigest Eco31I 38 Eco31I 110 Bso31I, BspTNI 

BsaJI CCNNGG FastDigest BsaJI 26 BseDI 94 

BsaMI FastDigest Mva1269I 51 Mva1269I 130 BsmI, PctI 

BsaWI WCCGGW 

BsaXI 5 Bsc4I CCNNNNNNNGG FastDigest BslI 28 BseLI 95 AfiI, BslI 

Bse118I RCCGGY FastDigest BsrFI 32 Cfr10I 105 BsrFI, BssAI 

Bse1I FastDigest BseNI 27 BseNI 96 BsrI, BsrSI 

Bse21I CCTNAGG FastDigest Bsu36I 34 Eco81I 113 AxyI, Bsu36I 

Bse3DI FastDigest BsrDI 32 BseMI 95 BsrDI 

Bse8I GATNNNNATC FastDigest BsaBI 25 BseJI 94 BsaBI 

BseAI TCCGGA FastDigest Kpn2Im 46 Kpn2I m 123 AccIIIm , Aor13HI, Bsp13I, BspEIm , MroIm BseBI CCWGG FastDigest MvaI 50 

[EcoRII 

MvaI 

116 

129 

[AjnI], Bst2UI, BstNI, BstOI, [Psp6I], 

[PspGI] 

BseCI ATCGAT FastDigest ClaI 34 Bsu15I 103 

BanIII, Bsa29I, BshVI, BspDI, BspXI, 

BsuTUI, ClaI 

BseDI CCNNGG FastDigest BsaJI 26 BseDI 94 

BseGI 

FastDigest BseGI, 

[FastDigest FokI 

26 

40 

BseGI 94 BstF5I, BtsCI, [FokI] 

BseJI GATNNNNATC FastDigest BsaBI 25 BseJI 94 BsaBI, Bse8I 

BseLI CCNNNNNNNGG FastDigest BslI 28 BseLI 95 AfiI, Bsc4I, BslI 

BseMI FastDigest BsrDI 32 BseMI 95 Bse3DI, BsrDI 

BseMII FastDigest BspCNI 31 BseMII 95 [BspCNI] 

BseNI FastDigest BseNI 27 BseNI 96 Bse1I, BsrI, BsrSI 

BsePI GCGCGC FastDigest BssHII 33 PauI 134 BssHII 

BseRI 

BseSI GKGCMC FastDigest Bme1580I 23 BseSI 96 BaeGI, BstSLI 

BseXI 

FastDigest BseXI, 


27 

21 

BseXI , 

Lsp1109I 

96 

124 

BbvI, BstV1I 

BseX3I CGGCCG FastDigest EagI 36 Eco52I 111 BstZI, EagI, EclXI 

BseYI [FastDigest PspFI 58 [GsaI] 


208 





BsgI 

Bsh1236I CGCG FastDigest Bsh1236I 27 Bsh1236I 97 AccII, BspFNI, BstFNI, BstUI, MvnI 

Bsh1285I CGRYCG FastDigest BsiEI 28 Bsh1285I 97 BsiEIm , BstMCI 

BshFI GGCC FastDigest HaeIII 42 BsuRI 103 BsnI, BspANI, HaeIII, PhoI 

BshNI GGYRCC FastDigest BanI 21 BshNI 97 AccB1I, BanI, BspT107I 

BshTI ACCGGT FastDigest AgeI 15 BshTI 98 AgeI, AsiGI, CspAIm , PinAI 

BshVI ATCGAT FastDigest ClaI 34 Bsu15I 103 

BanIII, Bsa29I, BseCI, BspDI, BspXI, 

BsuTUI, ClaI 

BsiEI CGRYCG FastDigest BsiEI m 28 Bsh1285I m 97 BsiEI, BstMCI 

BsiHKAI GWGCWC FastDigest Alw21I 17 Alw21I 86 Bbv12I 

BsiHKCI CYCGRG FastDigest AvaI 19 Eco88I 113 Ama87I, AvaI, [BmeT110I], BsoBI 

BsiSI CCGG 

FastDigest HpaIIm , 


44 

50 

HpaIIm , 

MspI 

121 

128 

HapIIm BsiWI CGTACG FastDigest BsiWI 28 Pfl23II 136 BsiWI, PspLI 

BslFI FastDigest BsmFI 29 FaqI 117 BsmFI 

BslI CCNNNNNNNGG FastDigest BslI 28 BseLI 95 AfiI, Bsc4I, BslI 

BsmAI FastDigest Alw26Im 17 Alw26I m 86 BstMAI 

BsmBI FastDigest BsmBI 29 Esp3I 117 BsmBI 

BsmFI FastDigest BsmFI 29 FaqI 117 BslFI, BsmFI 

BsmI FastDigest Mva1269I 51 Mva1269I 130 BsaMI, PctI 

BsnI GGCC FastDigest HaeIII 42 BsuRI 103 BshFI, BspANI, HaeIII, PhoI 

Bso31I FastDigest Eco31I 38 Eco31I 110 BsaI, BspTNI 

BsoBI CYCGRG FastDigest AvaI m 19 Eco88I m 113 Ama87I, AvaIm , [BmeT110I], BsiHKCI 

Bsp119I TTCGAA FastDigest Bsp119I 29 Bsp119I 99 

AsuIIm , Bpu14I, BspT104I, BstBI, 

Csp45I, NspV, SfuIm Bsp120I GGGCCC 

[FastDigest ApaI 


18 

30 

[ApaI 

Bsp120I 

87 

99 

PspOMI 

Bsp1286I GDGCHC FastDigest Bsp1286I 30 SduI 143 Bsp1286I, MhlI 

Bsp1407I TGTACA FastDigest Bsp1407I 30 Bsp1407I 100 BsrGI, BstAUI 

Bsp143I GATC 



46 

Bsp143I , 

MboIm 99 

126 

BfuCI, BssMI, [BstKTIm ], BstMBI, DpnIIm , 

Kzo9I, NdeIIm , Sau3AIm Bsp13I TCCGGA FastDigest Kpn2I 46 Kpn2I 123 AccIII, Aor13HI, BseAI, BspEI, MroI 

Bsp1720I GCTNAGC FastDigest BlpI 23 Bpu1102I 93 BlpI, CelII 

Bsp19I CCATGG FastDigest NcoI 52 NcoI 130 

Bsp68I TCGCGA FastDigest NruI 54 Bsp68I 98 BtuMI, NruI 

BspACI FastDigest AciI 14 SsiI 147 AciI 

BspANI GGCC FastDigest HaeIII 42 BsuRI 103 BshFI, BsnI, HaeIII, PhoI 

BspCNI 

[FastDigest BspCNI ] 

 

31 [BseMII 95 BspCNI 

BspDI ATCGAT FastDigest ClaI 34 Bsu15I 103 

BanIII, Bsa29I, BseCI, BshVI, BspXI, 

BsuTUI, ClaI 

BspEI TCCGGA FastDigest Kpn2Im 46 Kpn2I m 123 AccIIIm , Aor13HI, BseAIm , Bsp13I, MroIm BspFNI CGCG FastDigest Bsh1236I 27 Bsh1236I 97 AccII, BstFNI, BstUI, MvnI 

BspHI TCATGA FastDigest BspHI m 31 PagI m 134 BspHI, CciI, RcaI 

BspLI GGNNCC FastDigest NlaIV 53 BspLI 95 BmiI, NlaIVm , PspN4I 

BspMAI CTGCAG FastDigest PstI 58 PstI 138 

BspMI FastDigest BspMI 31 BveI 104 Acc36I, BfuAI, BspMI 

BspOI GCTAGC 

FastDigest BmtI , 

[FastDigest NheI 

24 

52 

BspOI , 

[NheI 

100 

131 

[AsuNHI], BmtI 

BspPI BspPI 101 AclWI, AlwI 

FastDigest SapI 61 LguI 124 PciSI, SapI 

BspT104I TTCGAA FastDigest Bsp119I 29 Bsp119I 99 AsuIIm , Bpu14I, BstBI, Csp45I, NspV, SfuIm BspT107I GGYRCC FastDigest BanI 21 BshNI 97 AccB1I, BanI 

BspTI CTTAAG FastDigest AflII 15 BspTI 101 AflII, BfrI, Bst98I, BstAFI, MspCI, Vha464I 

BspTNI FastDigest Eco31I 38 Eco31I 110 BsaI, Bso31I 

BspXI ATCGAT FastDigest ClaI 34 Bsu15I 103 

BanIII, Bsa29I, BseCI, BshVI, BspDI, 

BsuTUI, ClaI 

BsrBI FastDigest BsrBI m 32 MbiI m 125 AccBSI, BsrBI 

BsrDI FastDigest BsrDI 32 BseMI 95 Bse3DI, BsrDI 

BsrFI RCCGGY FastDigest BsrFI 32 Cfr10I 105 Bse118I, BsrFI, BssAIm BsrGI TGTACA FastDigest Bsp1407I 30 Bsp1407I 100 BstAUI 

BsrI FastDigest BseNI 27 BseNI 96 Bse1I, BsrSI 

BsrSI FastDigest BseNI 27 BseNI 96 Bse1I, BsrI 

BssAI RCCGGY FastDigest BsrFI m 32 Cfr10I m 105 Bse118I, BsrFIm BssECI CCNNGG FastDigest BsaJI 26 BseDI 94 

BssHII GCGCGC FastDigest BssHII 33 PauI 134 BsePI, BssHII 

BssKI CCNGG [FastDigest ScrFI 63 [Bme1390I 91 [BmrFI], BstSCI, [MspR9I], [ScrFI], StyD4I 






BssMI GATC 



62 

46 

Bsp143I , 

MboI 

99 

126 

BfuCI, [BstKTI], BstMBI, DpnII, Kzo9I, 

NdeII, Sau3AI 

BssNAI GTATAC FastDigest BstZ17I 33 Bst1107I 102 BstZ17I 

BssNI GRCGYC FastDigest BsaHI 26 Hin1I 119 AcyI, BsaHI, BstACI, Hsp92I 

BssSI BauI 88 Bst2BI 

BssT1I CCWWGG FastDigest StyI 67 Eco130I 114 EcoT14I, ErhI, StyI 

Bst1107I GTATAC FastDigest BstZ17I 33 Bst1107I 102 BssNAI, BstZ17I 

Bst2BI BauI 88 BssSI 

Bst2UI CCWGG FastDigest MvaI 50 

[EcoRIIm MvaI 

116 

129 

[AjnI], BseBI, BstNI, BstOI, [Psp6I], 

[PspGIm ] 

Bst4CI ACNGT FastDigest TaaI 67 TaaI 149 HpyCH4III 

Bst6I FastDigest EarI 37 Eam1104I 108 EarI 

Bst98I CTTAAG FastDigest AflII 15 BspTI 101 AflII, BfrI, BstAFI, MspCI, Vha464I 

BstACI GRCGYC FastDigest BsaHI 26 Hin1I 119 AcyI, BsaHI, BssNI, Hsp92I 

BstAFI CTTAAG FastDigest AflII 15 BspTI 101 AflII, BfrI, Bst98I, MspCI, Vha464I 

BstAPI GCANNNNNTGC 

BstAUI TGTACA FastDigest Bsp1407I 30 Bsp1407I 100 BsrGI 

BstBAI YACGTR FastDigest BsaAI 25 Ppu21I 137 BsaAI 

BstBI TTCGAA FastDigest Bsp119I 29 Bsp119I 99 

AsuIIm , Bpu14I, BspT104I, Csp45I, 

NspVm , SfuIm BstC8I GCNNGC Cac8I 

BstDEI CTNAG FastDigest DdeI 35 HpyF3I 122 DdeI 

BstDSI CCRYGG BtgI 

BstEII GGTNACC FastDigest Eco91I 38 Eco91I 113 BstPI, EcoO65I, PspEI 

BstENI CCTNNNNNAGG FastDigest EcoNI 38 XagI 152 EcoNI 

BstF5I 



26 

40 

BseGI 94 BtsCI, [FokI] 

BstFNI CGCG FastDigest Bsh1236I 27 Bsh1236I 97 AccII, BspFNI, BstUI, MvnI 

BstH2I RGCGCY FastDigest HaeII 43 HaeII 

BstHHI GCGC 



42 

44 

HhaI, 

[Hin6I 

119 

120 

AspLEI, CfoI, [HinP1I], [HspAI] 

BstKTI GATC 

[FastDigest Sau3AI m [FastDigest MboI 

62 

46 

[Bsp143I m [MboI 

99 

126 

[BfuCIm ], [BssMI], [BstMBI], [DpnII], 

[Kzo9Im ], [NdeIIm ], [Sau3AIm ] 

BstMAI FastDigest Alw26I 17 Alw26I 86 BsmAI 

BstMBI GATC 



62 

46 

Bsp143I , 

MboI 

99 

126 

BfuCI, BssMI, [BstKTI], DpnII, Kzo9I, 

NdeII, Sau3AI 

BstMCI CGRYCG FastDigest BsiEI 28 Bsh1285I 97 BsiEI 

BstMWI GCNNNNNNNGC FastDigest HpyF10VI 45 HpyF10VI 123 MwoI 

BstNI CCWGG FastDigest MvaI 50 


116 

129 

[AjnI], BseBI, Bst2UI, BstOI, [Psp6I], 

[PspGIm ] 

BstNSI RCATGY FastDigest NspI 55 XceI 153 NspI 

BstOI CCWGG FastDigest MvaI 50 


116 

129 

[AjnI], BseBI, Bst2UI, BstNI, [Psp6I], 

[PspGIm ] 

BstPAI GACNNNNGTC FastDigest PshAI 57 BoxI 92 PshAI 

BstPI GGTNACC FastDigest Eco91I 38 Eco91I 113 BstEII, EcoO65I, PspEI 

BstSCI CCNGG [FastDigest ScrFI 63 [Bme1390I 91 [BmrFI], BssKI, [MspR9I], [ScrFI], StyD4I 

BstSFI CTRYAG FastDigest SfcI 64 BfmI 90 SfcI 

BstSLI GKGCMC FastDigest Bme1580I 23 BseSI 96 BaeGI 

BstSNI TACGTA FastDigest SnaBI 65 Eco105I 114 SnaBI 

BstUI CGCG FastDigest Bsh1236I 27 Bsh1236I 97 AccII, BspFNI, BstFNI, MvnI 

BstV1I 



21 

27 

Lsp1109I , 

BseXI 

124 

96 

BbvI 

BstV2I FastDigest BbsI 21 BpiI 92 BbsI, BpuAI 

BstX2I RGATCY FastDigest PsuI 58 PsuI 139 BstYI, MflI, XhoII 

BstXI CCANNNNNNTGG FastDigest BstXI 33 BstXI 102 

BstYI RGATCY FastDigest PsuI 58 PsuI 139 BstX2I, MflIm , XhoII 

BstZ17I GTATAC FastDigest BstZ17I 33 Bst1107I 102 BssNAI, BstZ17I 

BstZI CGGCCG FastDigest EagI 36 Eco52I 111 BseX3I, EagI, EclXI 

Bsu15I ATCGAT FastDigest ClaI 34 Bsu15I 103 


BspXI, BsuTUI, ClaI 

Bsu36I CCTNAGG FastDigest Bsu36I 34 Eco81I 113 AxyI, Bse21I, Bsu36I 

BsuRI GGCC FastDigest HaeIII 42 BsuRI 103 BshFI, BsnI, BspANI, HaeIII, PhoI 

BsuTUI ATCGAT FastDigest ClaI 34 Bsu15I 103 


BspXI, ClaI 

BtgI CCRYGG BstDSI 

BtgZI 

BtrI AjiI 84 BmgBI 

 


210 



BtsCI 




26 

40 

BseGI 94 BstF5I, [FokI] 



BtsI 

BtuMI TCGCGA FastDigest NruI 54 Bsp68I 98 NruI 

BveI FastDigest BspMI 31 BveI 104 Acc36I, BfuAI, BspMI 

Cac8I GCNNGC BstC8I 

CaiI CAGNNNCTG FastDigest AlwNI 17 CaiI 104 AlwNI 

CciI TCATGA FastDigest BspHI 31 PagI 134 BspHI, RcaI 

CciNI GCGGCCGC FastDigest NotI 54 NotI 132 

CelII GCTNAGC FastDigest BlpI 23 Bpu1102I 93 BlpI, Bsp1720I 

CfoI GCGC 



42 

44 

HhaI, 

[Hin6I 

119 

120 

AspLEI, BstHHI, [HinP1I], [HspAI] 

Cfr10I RCCGGY FastDigest BsrFI 32 Cfr10I 105 Bse118I, BsrFI, BssAIm Cfr13I GGNCC FastDigest Sau96I 62 Cfr13I 105 AspS9I, [BmgT120I], PspPI, Sau96I 

Cfr42I CCGCGG Cfr42I 106 KspI, SacII, Sfr303I, SgrBI, SstII 

Cfr9I CCCGGG [FastDigest SmaIm 65 

Cfr9I , 

[SmaIm 105 

146 

TspMIm , XmaCI, XmaIm CfrI YGGCCR AcoI, EaeI 

ClaI ATCGAT FastDigest ClaI 34 Bsu15I 103 


BspXI, BsuTUI, ClaI 

CpoI CGGWCCG FastDigest RsrII 60 CpoI 106 CspI, Rsr2I, RsrII 

CseI FastDigest HgaI 42 CseI 106 HgaI 

Csp45I TTCGAA FastDigest Bsp119I 29 Bsp119I 99 

AsuII, Bpu14I, BspT104I, BstBI, 

NspVm , SfuI 

Csp6I GTAC 

FastDigest Csp6I, 

[FastDigest RsaIm 34 

60 

Csp6I , 

[RsaIm 107 

140 

[AfaIm 

CspAI ACCGGT FastDigest AgeI m 15 BshTI m 98 AgeIm , AsiGI, PinAI 

CspCI 5 CspI CGGWCCG FastDigest RsrII 60 CpoI 106 Rsr2I, RsrII 

CviAII CATG [FastDigest NlaIII 53 [Hin1II 120 [FaeI], [FatIm ][Hsp92II], [NlaIII] 

RGCY CviKI-1 

CviKI-1 RGCY 

GTAC 


[FastDigest RsaIm 34 

60 

Csp6I , 

[RsaIm 107 

140 

[AfaIm ], RsaNI 

DdeI CTNAG FastDigest DdeI 35 HpyF3I 122 BstDEI, DdeI 

DinI GGCGCC FastDigest EheI 116 

EheI , 

[SspDI 

116 

148 

[BbeI], EgeI, [KasI], [Mly113I], [NarI], 

SfoI 

DpnI Gm6ATC FastDigest DpnI 35 DpnI 107 MalI 

DpnII GATC 

FastDigest Sau3AI m , 


46 

[Bsp143I m ], 

MboIm 99 

126 

BfuCIm , BssMI, [BstKTIm ], BstMBI, 

Kzo9Im , NdeII, Sau3AIm DraI TTTAAA FastDigest DraI 35 DraI 108 

DraII RGGNCCY FastDigest EcoO109I 39 EcoO109I 115 

DraIII CACNNNGTG FastDigest DraIII 36 AdeI 83 DraIII 

DrdI GACNNNNNNGTC FastDigest DrdI 36 DrdI, DseDI 

DriI GACNNNNNGTC FastDigest Eam1105I 37 Eam1105I 108 AhdI, AspEI, BmeRI, EclHKI 

DseDI GACNNNNNNGTC FastDigest DrdI 36 DrdI 

EaeI YGGCCR AcoI 

EagI CGGCCG FastDigest EagI 36 Eco52I 111 BseX3I, BstZI, EagI, EclXI 

Eam1104I FastDigest EarI 37 Eam1104I 108 Bst6I, EarI 

Eam1105I GACNNNNNGTC FastDigest Eam1105I 37 Eam1105I 108 AhdI, AspEI, BmeRI, DriI, EclHKI 

EarI FastDigest EarI 37 Eam1104I 108 Bst6I, EarI 

EciI 

Ecl136II GAGCTC 

FastDigest Ecl136II, 

[FastDigest SacIm 37 

60 

Ecl136II , 

[SacIm 109 

141 

Eco53kIm , EcoICRI, [Psp124BI], [SstIm ] 

EclHKI GACNNNNNGTC FastDigest Eam1105I 37 Eam1105I 108 AhdI, AspEI, BmeRI, DriI 

EclXI CGGCCG FastDigest EagI 36 Eco52I 111 BseX3I, BstZI, EagI 

Eco105I TACGTA FastDigest SnaBI 65 Eco105I 114 BstSNI, SnaBI 

Eco130I CCWWGG FastDigest StyI 67 Eco130I 114 BssT1I, EcoT14I, ErhI, StyI 

Eco147I AGGCCT FastDigest StuI 66 Eco147I 114 AatI, PceI, SseBI, StuI 

Eco24I GRGCYC Eco24I 109 BanII, EcoT38I, FriOI 

Eco31I FastDigest Eco31I 38 Eco31I 110 BsaI, Bso31I, BspTNI 

Eco32I GATATC FastDigest EcoRV 39 Eco32I 110 EcoRV 

Eco47I GGWCC FastDigest AvaII 20 Eco47I 110 AvaII, Bme18I, SinI, VpaK11BI 

Eco47III AGCGCT FastDigest AfeI 15 Eco47III 111 AfeI, Aor51HI 

Eco52I CGGCCG FastDigest EagI 36 Eco52I 111 BseX3I, BstZI, EagI, EclXI 

Eco53kI GAGCTC 

FastDigest Ecl136II m , 

[FastDigest SacI m 

37 

60 

Ecl136II m , 

[SacI m 

109 

141 

EcoICRI, [Psp124BI], [SstI]





Eco57I FastDigest AcuI 14 Eco57I 112 AcuI 

Eco57MI 

Eco72I CACGTG FastDigest PmlI 56 Eco72I 112 AcvI, BbrPI, PmaCI, PmlI, PspCI 

Eco81I CCTNAGG FastDigest Bsu36I 34 Eco81I 113 AxyI, Bse21I, Bsu36I 

Eco88I CYCGRG FastDigest AvaI 19 Eco88I 113 

Ama87I, AvaI 

 

m , [BmeT110Im ], BsiHKCI, 

BsoBIm Eco91I GGTNACC FastDigest Eco91I 38 Eco91I 113 BstEII, BstPI, EcoO65I, PspEI 

EcoICRI GAGCTC 

FastDigest Ecl136II, 

[FastDigest SacI 

37 

60 

Ecl136II , 

[SacI 

109 

141 

Eco53kI, [Psp124BI], [SstI] 

EcoNI CCTNNNNNAGG FastDigest EcoNI 38 XagI 152 BstENI, EcoNI 

EcoO109I RGGNCCY FastDigest EcoO109I 39 EcoO109I 115 DraII 

EcoO65I GGTNACC FastDigest Eco91I 38 Eco91I 113 BstEII, BstPI, PspEI 

EcoP15I 

EcoRI GAATTC FastDigest EcoRI 39 EcoRI 115 

EcoRII CCWGG [FastDigest MvaIm 50 

EcoRII, 

[MvaI m 116 

129 

AjnIm , [BseBI], [Bst2UIm ], [BstNIm ], 

[BstOIm ], Psp6I, PspGI 

EcoRV GATATC FastDigest EcoRV 39 Eco32I 110 EcoRV 

EcoT14I CCWWGG FastDigest StyI 67 Eco130I 114 BssT1I, ErhI, StyI 

EcoT22I ATGCAT FastDigest NsiI 54 Mph1103I 128 NsiIm , Zsp2I 

EcoT38I GRGCYC Eco24I 109 BanII, FriOI 

EgeI GGCGCC FastDigest EheI 40 

EheI , 

[SspDI 

116 

148 

[BbeI], DinI, [KasI], [Mly113I], [NarI], 

SfoI 

EheI GGCGCC FastDigest EheI 40 

EheI , 

[SspDI 

116 

148 

[BbeI], DinI, EgeI, [KasI], [Mly113I], 

[NarI], SfoIm ErhI CCWWGG FastDigest StyI 67 Eco130I 114 BssT1I, EcoT14I, StyI 

Esp3I FastDigest BsmBI 29 Esp3I 117 BsmBI 

FaeI CATG FastDigest NlaIII 53 Hin1II 120 [CviAII], [FatI], Hsp92II, NlaIII 

FaiI YATR 

FaqI FastDigest BsmFI 29 FaqI 117 BslFI, BsmFI 

FalI 5 FatI CATG [FastDigest NlaIII 53 [Hin1II 120 [CviAIIm ], [FaeI], [Hsp92II], [NlaIIIm ] 

FauI 

FauNDI CATATG FastDigest NdeI 52 NdeI 131 

FbaI TGATCA FastDigest BclI 22 BclI 89 Ksp22I 

FblI GTMKAC FastDigest AccI 13 XmiI 154 AccI 

Fnu4HI GCNGC FastDigest Fnu4HI 40 SatI 142 Fnu4HI, Fsp4HI, ItaIm FokI 

[FastDigest BseGI 

FastDigest FokI 

26 

40 

[BseGI 94 [BstF5I], [BtsCI], FokI 

FriOI GRGCYC Eco24I 109 BanII, EcoT38I 

FseI GGCCGGCC RigI 

Fsp4HI GCNGC FastDigest Fnu4HI 40 SatI 142 Fnu4HI, ItaIm FspAI RTGCGCAY FastDigest FspAI 41 FspAI 118 

FspBI CTAG FastDigest BfaI 22 FspBI 118 BfaI, MaeI, XspI 

FspI TGCGCA FastDigest FspI 41 NsbI 132 Acc16I, AviII, FspI 

GlaI GCGC 

GluI GCNGC BisI, [BlsI] 

GsaI FastDigest PspFI 58 [BseYI] 

GsuI FastDigest BpmI 24 GsuI 118 BpmIm HaeII RGCGCY FastDigest HaeII 41 BstH2I, HaeII 

HaeIII GGCC FastDigest HaeIII 42 BsuRI 103 BshFI, BsnI, BspANI, HaeIII, PhoI 

HapII CCGG 

FastDigest HpaII, 

FastDigest MspIm 44 

50 

HpaII, 

MspI m 121 

128 

BsiSIm HgaI FastDigest HgaI 42 CseI 106 HgaI 

HhaI GCGC 



42 

44 

HhaI, 

[Hin6I 

119 

120 

AspLEI, BstHHI, CfoI, [HinP1I], [HspAI] 

Hin1I GRCGYC FastDigest BsaHI 26 Hin1I 119 AcyI, BsaHIm , BssNI, BstACI, Hsp92I 

Hin1II CATG FastDigest NlaIII 53 Hin1II 120 [CviAII], FaeI, [FatI], Hsp92II, NlaIII 

Hin4I 5 Hin6I GCGC 

[FastDigest HhaI FastDigest 

HinP1I 

42 

44 

[HhaI 

Hin6I 

119 

120 

[AspLEI], [BstHHI], [CfoI], HinP1I, HspAI 

HincII GTYRAC FastDigest HincII 43 HincII 120 HindII 

HindII GTYRAC FastDigest HincII 43 HincII 120 

HindIII AAGCTT FastDigest HindIII 43 HindIII 121 

HinfI GANTC FastDigest HinfI 43 HinfI 121 

HinP1I GCGC 


HinP1I 

42 

44 

[HhaI 

Hin6I 

119 

120 

[AspLEI], [BstHHI], [CfoI], HinP1I, HspAI 

HpaI GTTAAC FastDigest HpaI m 44 KspAI m 124 HpaI 


212 





HpaII CCGG 

FastDigest HpaII, 

FastDigest MspIm 44 

50 

HpaII, 

MspI m 121 

128 

BsiSIm , HapII 

HphI HphI 122 AsuHPI 

Hpy166II GTNNAC FastDigest Hpy8I 45 Hpy8I 122 

Hpy188I TCNGA 

Hpy188III TCNNGA 

Hpy8I GTNNAC FastDigest Hpy8I 45 Hpy8I 122 Hpy166II 

Hpy99I CGWCG 

HpyAV 

HpyCH4III ACNGT FastDigest TaaI 67 TaaI 149 Bst4CI 

HpyCH4IV ACGT [FastDigest TaiIm 68 [TaiI m 149 MaeII 

HpyCH4V TGCA 

HpyF10VI GCNNNNNNNGC FastDigest HpyF10VI 45 HpyF10VI 123 BstMWI, MwoIm HpyF3I CTNAG FastDigest DdeI 35 HpyF3I 122 BstDEI, DdeI 

Hsp92I GRCGYC FastDigest BsaHI 26 Hin1I 119 AcyI, BsaHI, BssNI, BstACI 

Hsp92II CATG FastDigest NlaIII 53 Hin1II 120 [CviAII], FaeI, [FatI], NlaIII 

HspAI GCGC 


HinP1I 

42 

44 

[HhaI 

Hin6I 

119 

120 

[AspLEI], [BstHHI], [CfoI], HinP1I 

ItaI GCNGC FastDigest Fnu4HI m 40 SatI m 142 Fnu4HIm , Fsp4HIm KasI GGCGCC [FastDigest EheI 40 

[EheI 

SspDI 

116 

148 

[BbeI], [DinI], [EgeI], [Mly113I], [NarI], 

[SfoIm ] 

Kpn2I TCCGGA FastDigest Kpn2I 46 Kpn2I 123 

AccIIIm , Aor13HI, BseAIm , Bsp13I, 

BspEIm , MroI 

KpnI GGTACC 

[FastDigest Acc65Im FastDigest KpnI 

13 

45 

[Acc65I m KpnI 

83 

123 

[Asp718Im ] 

Ksp22I TGATCA FastDigest BclI 22 BclI 89 FbaI 

KspAI GTTAAC FastDigest HpaI 44 KspAI 124 HpaI 

KspI CCGCGG Cfr42I 106 SacII, Sfr303I, SgrBI, SstII 

Kzo9I GATC 



46 

Bsp143I , 

MboIm 99 

126 

BfuCI, BssMI, [BstKTIm ], BstMBI, 

DpnIIm , NdeIIm , Sau3AI 

LguI FastDigest SapI 61 LguI 124 

Lsp1109I 



21 

27 

Lsp1109I , 

BseXI 

124 

96 

BbvI, BstV1I 

LweI FastDigest SfaNI 64 LweI 125 SfaNI 

MabI ACCWGGT FastDigest SexAI 63 SexAIm MaeI CTAG FastDigest BfaI 22 FspBI 118 BfaI, XspI 

MaeII ACGT [FastDigest TaiI 68 [TaiI 149 HpyCH4IV 

MaeIII GTNAC 

MalI GATC FastDigest DpnI 35 DpnI 107 

MauBI CGCGCGCG FastDigest MauBI 46 MauBI 125 

MbiI FastDigest BsrBI 32 MbiI 125 AccBSI, BsrBIm MboI GATC 



62 

46 

Bsp143I m , 

MboI 

99 

126 

BfuCIm , BssMI, [BstKTI], BstMBI, 

DpnIIm , Kzo9Im , NdeIIm , Sau3AIm MboII FastDigest MboII 47 MboII 126 

MfeI CAATTG FastDigest MfeI 47 MunI 129 MfeI 

MflI RGATCY FastDigest PsuIm 58 PsuI m 139 BstX2I, BstYIm , XhoIIm MhlI GDGCHC FastDigest Bsp1286I 30 SduI 143 Bsp1286I 

MlsI TGGCCA FastDigest MscI 49 MlsI 127 BalI, MluNI, MscI, Msp20I 

MluI ACGCGT FastDigest MluI 47 MluI 127 

MluNI TGGCCA FastDigest MscI 49 MlsI 127 BalI, MscI, Msp20I 

Mly113I GGCGCC [FastDigest EheI 40 

[EheI 

[SspDI 

116 

148 

[BbeI], [DinI], [EgeI], [KasI], NarI, [SfoI] 

MlyI FastDigest MlyI 48 SchI 143 MlyI, [PleIm ], [PpsI] 

MmeI 

MnlI FastDigest MnlI 48 MnlI 127 

Mph1103I ATGCAT FastDigest NsiI 54 Mph1103I 128 EcoT22I, NsiI, Zsp2I 

MreI CGCCGGCG FastDigest MreI 48 MreI 128 

MroI TCCGGA FastDigest Kpn2I 46 Kpn2I 123 AccIIIm , Aor13HI, BseAIm , Bsp13I, BspEIm MroNI GCCGGC [FastDigest NaeI 51 [PdiI 135 [NaeI], NgoMIV 

MroXI GAANNNNTTC FastDigest PdmI 55 PdmI 135 Asp700I, XmnI 

MscI TGGCCA FastDigest MscI 49 MlsI 127 BalI, MscI, MluNI, Msp20I 

MseI TTAA 

FastDigest MseI , 


49 

69 

Tru1I 151 MseI, Tru9I 

MslI CAYNNNNRTG FastDigest MslI 49 RseI 140 MslI, SmiMI 

Msp20I TGGCCA FastDigest MscI 49 MlsI 127 BalI, MluNI, MscI 

MspA1I CMGCKG 

MspCI CTTAAG FastDigest AflII 15 BspTI 101 AflII, BfrI, Bst98I, BstAFI, Vha464I 






MspI CCGG 

FastDigest HpaIIm , 


44 

50 

HpaIIm , 

MspI 

121 

128 

BsiSI, HapIIm MspR9I CCNGG FastDigest ScrFI 63 Bme1390I 91 BmrFI, [BssKI], [BstSCI], ScrFI, [StyD4I] 

MssI GTTTAAAC FastDigest MssI 50 MssI 129 PmeIm MunI CAATTG FastDigest MfeI 47 MunI 129 MfeI 

Mva1269I FastDigest Mva1269I 51 Mva1269I 130 BsaMI, BsmI, PctI 

MvaI CCWGG FastDigest MvaI 50 


116 

129 

[AjnI], BseBI, Bst2UI, BstNI, BstOI, 

[Psp6I], [PspGIm ] 

MvnI CGCG FastDigest Bsh1236I 27 Bsh1236I 97 AccII, BspFNI, BstFNI, BstUI 

MvrI CGATCG FastDigest PvuI 59 PvuI 139 BpvUI, Ple19I 

MwoI GCNNNNNNNGC FastDigest HpyF10VIm 123 HpyF10VI m 123 BstMWI 

NaeI GCCGGC FastDigest NaeI 51 PdiI 135 [MroNI], NaeI, [NgoMIV] 

NarI GGCGCC [FastDigest EheI 40 

[EheI 

[SspDI 

116 

148 

[BbeI], [DinI], [EgeI], [KasI], Mly113I, 

[SfoIm ] 

NciI CCSGG FastDigest NciI 51 BcnI 89 AsuC2I, BpuMI, NciI 

NcoI CCATGG FastDigest NcoI 52 NcoI 130 Bsp19I 

NdeI CATATG FastDigest NdeI 52 NdeI 131 FauNDI 

NdeII GATC 



46 

Bsp143I m , 

MboIm 99 

126 


DpnII, Kzo9Im , Sau3AIm NgoMIV GCCGGC [FastDigest NaeI 51 [PdiI 135 MroNI, [NaeI] 

NheI GCTAGC 

FastDigest NheI, 

[FastDigest BmtI 

52 

24 

NheI, 

[BspOI 

131 

100 

AsuNHI, [BmtI] m 

NlaIII CATG FastDigest NlaIII 53 Hin1II 120 [CviAII], FaeI, [FatIm ], Hsp92II, NlaIII 

NlaIV GGNNCC FastDigest NlaIV m 53 BspLI m 100 BmiI, NlaIV, PspN4I 

NmeAIII 

NmuCI GTSAC FastDigest NmuCI 53 NmuCI 132 Tsp45I 

NotI GCGGCCGC FastDigest NotI 54 NotI 132 CciNI 

NruI TCGCGA FastDigest NruI 54 Bsp68I 98 BtuMI, NruI 

NsbI TGCGCA FastDigest FspI 41 NsbI 132 Acc16I, AviII, FspI 

NsiI ATGCAT FastDigest NsiI 54 Mph1103I 128 EcoT22Im , NsiI, Zsp2I 

NspI RCATGY FastDigest NspI 55 XceI 153 BstNSI, NspI 

NspV TTCGAA FastDigest Bsp119I 29 Bsp119I 99 

AsuIIm , Bpu14I, BspT104I, BstBIm , 

Csp45Im , SfuIm OliI CACNNNNGTG FastDigest AleI 16 OliI 133 AleI 

PacI TTAATTAA FastDigest PacI 55 PacI 133 

PaeI GCATGC FastDigest SphI 66 PaeI 133 BbuI, SphI 

PaeR7I CTCGAG FastDigest XhoI 71 XhoI 154 Sfr274I, SlaI, StrI, TliI 

PagI TCATGA FastDigest BspHI 31 PagI 134 CciI, BspHIm , RcaI 

PalAI GGCGCGCC FastDigest AscI 18 SgsI 145 AscI 

PasI CCCWGGG PasI 134 

PauI GCGCGC FastDigest BssHII 33 PauI 134 BsePI, BssHII 

PceI AGGCCT FastDigest StuI 66 Eco147I 114 AatI, SseBI, StuI 

PciI ACATGT PscI 137 

PciSI FastDigest SapI 61 LguI 124 

PctI FastDigest Mva1269I 51 Mva1269I 130 BsaMI, BsmI 

PdiI GCCGGC FastDigest NaeI 51 PdiI 135 [MroNI], NaeI, [NgoMIV] 

PdmI GAANNNNTTC FastDigest PdmI 55 PdmI 135 Asp700I, MroXI, XmnIm PfeI GAWTC FastDigest TfiI 69 PfeI 135 TfiI 

Pfl23II GGTACG FastDigest BsiWI 28 Pfl23II 136 BsiWI, PspLI 

PflFI GACNNNGTC FastDigest PsyI 59 PsyI 139 AspI, Tth111I 

PflMI CCANNNNNTGG FastDigest PflMI 56 Van91I 152 AccB7I, BasI, PflMI 

PfoI TCCNGGA FastDigest PfoI 56 PfoI 136 

PhoI GGCC FastDigest HaeIII 42 BsuRI 103 BshFI, BsnI, BspANI, HaeIII 

PinAI ACCGGT FastDigest AgeI 15 BshTI 101 AgeI, AsiGI, CspAI 

Ple19I CGATCG FastDigest PvuI 59 PvuI 139 BpvUI, MvrI 

PleI [FastDigest MlyI 48 [SchI 143 [MlyIm ], PpsI 

PmaCI CACGTG FastDigest PmlI 56 Eco72I 112 AcvI, BbrPI, PmlI, PspCI 

PmeI GTTTAAAC FastDigest MssIm 50 MssI m 129 

PmlI CACGTG FastDigest PmlI 56 Eco72I 112 AcvI, BbrPI, PmaCI, PmlI, PspCI 

PpiI 5 PpsI [FastDigest MlyI 48 [SchI 143 [MlyI], PleI 

Ppu21I YACGTR FastDigest BsaAI 25 Ppu21I 137 BsaAI, BstBAI 

PpuMI RGGWCCY FastDigest PpuMI 57 Psp5II 138 PpuMI, PspPPI 

PscI ACATGT PscI 137 PciI 

PshAI GACNNNNGTC FastDigest PshAI 57 BoxI 92 BstPAI, PshAI 

PshBI ATTAAT FastDigest AseI 19 VspI 152 AseI 

 


214 





PsiI TTATAA FastDigest PsiI 57 AanI 82 PsiI 

Psp124BI GAGCTC 

[FastDigest Ecl136II 


37 

60 

[Ecl136II 

SacI 

109 

141 

[Eco53kI], [EcoICRI], SstI 

Psp1406I AACGTT FastDigest AclI 14 Psp1406I 138 AclI 

Psp5II RGGWCCY FastDigest PpuMI 57 Psp5II 138 PpuMI, PspPPI 

Psp6I CCWGG [FastDigest MvaI 50 

EcoRII, 

[MvaI 

116 

129 

AjnI, [BseBI], [Bst2UI], [BstNI], [BstOI], 

PspGI 

PspCI CACGTG FastDigest PmlI 56 Eco72I 112 AcvI, BbrPI, PmaCI, PmlI 

PspEI GGTNACC FastDigest Eco91I 38 Eco91I 113 BstEII, BstPI, EcoO65I 

PspFI FastDigest PspFI 58 GsaI, [BseYI] 

PspGI CCWGG [FastDigest MvaIm 129 

EcoRII, 

[MvaI m 116 

129 

AjnIm , [BseBI], [Bst2UIm ], [BstNIm ], 

[BstOIm ], Psp6I 

PspLI CGTACG FastDigest BsiWI 28 Pfl23II 136 BsiWI 

PspN4I GGNNCC FastDigest NlaIV 53 BspLI 100 BmiI, NlaIV 

PspOMI GGGCCC 

[FastDigest ApaI 


18 

30 

[ApaI 

Bsp120I 

87 

99 

PspPI GGNCC FastDigest Sau96I 62 Cfr13I 105 AspS9I, [BmgT120I], Sau96I 

PspPPI RGGWCCY FastDigest PpuMI 57 Psp5II 138 PpuMI 

PspXI VCTCGAGB 

PsrI 6 PstI CTGCAG FastDigest PstI 58 PstI 138 BspMAI 

PsuI RGATCY FastDigest PsuI 58 PsuI 139 BstX2I, BstYI, MflIm , XhoII 

PsyI GACNNNGTC FastDigest PsyI 59 PsyI 139 AspI, PflFI, Tth111I 

PvuI CGATCG FastDigest PvuI 59 PvuI 139 BpvUI, MvrI, Ple19I 

PvuII CAGCTG FastDigest PvuII 59 PvuII 140 

RcaI TCATGA FastDigest BspHI 31 PagI 134 BspHI, CciI 

RgaI GCGATCGC FastDigest AsiSI 19 SfaAI 144 AsiSI, SgfI 

RigI GGCCGGCC FseI 

RsaI GTAC 

[FastDigest Csp6Im FastDigest RsaI 

34 

60 

[Csp6I m RsaI 

107 

140 

m ], [RsaNI] 

RsaNI GTAC 


[FastDigest RsaI 

34 

60 

Csp6I , 

[RsaI 

107 

140 

 

RseI CAYNNNNRTG FastDigest MslI 49 RseI 140 MslI, SmiMI 

Rsr2I CGGWCCG FastDigest RsrII 60 CpoI 106 CspI, RsrII 

RsrII CGGWCCG FastDigest RsrII 60 CpoI 106 CspI, Rsr2I, RsrII 

SacI GAGCTC 

[FastDigest Ecl136IIm FastDigest SacI 

37 

60 

[Ecl136II m SacI 

109 

141 

[Eco53kIm ], [EcoICRI], Psp124BI, SstI 

SacII CCGCGG Cfr42I 106 KspI, Sfr303I, SgrBI, SstII 

SalI GTCGAC FastDigest SalI 61 SalI 141 

SanDI GGGWCCC FastDigest SanDI 61 SanDI 

SapI FastDigest SapI 61 LguI 124 

SatI GCNGC FastDigest Fnu4HI 40 SatI 142 Fnu4HI, Fsp4HI, ItaIm Sau3AI GATC 



46 

Bsp143I , 

MboIm 99 

126 


DpnIIm , Kzo9I, NdeIIm Sau96I GGNCC FastDigest Sau96I 62 Cfr13I 105 AspS9I, [BmgT120I], PspPI, Sau96I 

SbfI CCTGCAGG FastDigest SbfI 62 SdaI 143 SbfI, Sse8387I 

ScaI AGTACT FastDigest ScaI 63 ScaI 142 AssI, BmcAI, ZrmI 

SchI FastDigest MlyI 48 SchI 143 MlyI, [PleI], [PpsI], SchI 

ScrFI CCNGG FastDigest ScrFI 63 Bme1390I 91 

BmrFI, [BssKI], [BstSCI], MspR9I, ScrFI, 

[StyD4I] 

SdaI CCTGCAGG FastDigest SbfI 62 SdaI 143 SbfI, Sse8387I 

SduI GDGCHC FastDigest Bsp1286I 30 SduI 143 Bsp1286I, MhlI 

SetI ASST 

SexAI ACCWGGT FastDigest SexAI m 63 MabI m , SexAI 

SfaAI GCGATCGC FastDigest AsiSI 19 SfaAI 144 AsiSI, RgaI, SgfI 

SfaNI FastDigest SfaNI 64 LweI 125 

SfcI CTRYAG FastDigest SfcI 64 BfmI 90 BstSFI, SfcI 

SfiI GGCCNNNNNGGCC FastDigest SfiI 64 SfiI 144 

SfoI GGCGCC FastDigest EheIm 40 

EheI m , 

[SspDI 

116 

148 

[BbeIm ], DinI, EgeI, [KasIm ], [Mly113I], 

[NarIm ] 

Sfr274I CTCGAG FastDigest XhoI 71 XhoI 154 PaeR7I, SlaI, StrI, TliI 

Sfr303I CCGCGG Cfr42I 106 KspI, SacII, SgrBI, SstII 

SgeI SgeI 145 

SfuI TTCGAA FastDigest Bsp119Im 29 Bsp119I m 99 

AsuII, Bpu14I, BspT104Im , BstBIm , 

Csp45I, NspVm SgfI GCGATCGC FastDigest AsiSI 19 SfaAI 144 AsiSI, RgaI 

SgrAI CRCCGGYG 






SgrBI CCGCGG Cfr42I 106 KspI, SacII, Sfr303I, SstII 

SgrDI CGTCGACG SgrDI 145 

SgsI GGCGCGCC FastDigest AscI 18 SgsI 145 AscI, PalAI 

SinI GGWCC FastDigest AvaII 20 Eco47I 110 AvaII, Bme18I, VpaK11BI 

SlaI CTCGAG FastDigest XhoI 71 XhoI 154 PaeR7I, Sfr274I, StrI, TliI 

SmaI CCCGGG FastDigest SmaI 65 

[Cfr9I m SmaI 

105 

146 

[TspMI], [XmaCI], [XmaIm ] 

SmiI ATTTAAAT FastDigest SwaI 67 SmiI 146 SwaI 

SmiMI CAYNNNNRTG FastDigest MslI 49 RseI 140 MslI 

SmlI CTYRAG SmoI 147 

SmoI CTYRAG SmoI 147 SmlI 

SmuI FauI 

SnaBI TACGTA FastDigest SnaBI 65 Eco105I 114 BstSNI, SnaBI 

SpeI ACTAGT FastDigest SpeI 65 BcuI 89 AhlI, SpeI 

SphI GCATGC FastDigest SphI 66 PaeI 133 BbuI, SphI 

SrfI GCCCGGGC 

Sse8387I CCTGCAGG FastDigest SbfI 62 SdaI 143 SbfI 

Sse9I AATT FastDigest Tsp509I 70 TasI 150 Tsp509I, TspEI 

SseBI AGGCCT FastDigest StuI 66 Eco147I 114 AatI, PceI, StuI 

SsiI FastDigest AciI 14 SsiI 147 AciI, BspACI 

SspDI GGCGCC [FastDigest EheI 40 

[EheI 

SspDI 

116 

148 

[BbeI], [DinI], [EgeI], KasI, [Mly113I], 

[NarI], [SfoI] 

SspI AATATT FastDigest SspI 66 SspI 148 

SstI GAGCTC 

[FastDigest Ecl136IIm FastDigest SacI 

37 

60 

[Ecl136II m SacI 

109 

141 

[Eco53kI], [EcoICRI], Psp124BI 

SstII CCGCGG Cfr42I 106 KspI, SacII, Sfr303I, SgrBI 

StrI CTCGAG FastDigest XhoI 71 XhoI 154 PaeR7I, Sfr274I, SlaI, TliI 

StuI AGGCCT FastDigest StuI 66 Eco147I 114 AatI, PceI, SseBI, StuI 

StyD4I CCNGG [FastDigest ScrFI 63 [Bme1390I 91 [BmrFI], BssKI, BstSCI, [MspR9I], [ScrFI] 

StyI CCWWGG FastDigest StyI 67 Eco130I 114 BssT1I, EcoT14I, ErhI, StyI 

SwaI ATTTAAAT FastDigest SwaI 67 SmiI 146 SwaI 

TaaI ACNGT FastDigest TaaI 149 TaaI 149 Bst4CI, HpyCH4III 

TaiI ACGT FastDigest TaiI 68 TaiI 149 [HpyCH4IVm ], [MaeII] 

TaqI TCGA FastDigest TaqI 68 TaqI 149 

TaqII 

, 

 

TasI AATT FastDigest Tsp509I 70 TasI 150 Sse9I, Tsp509I, TspEI 

TatI WGTACW FastDigest TatI 68 TatI 150 

TauI GCSGC FastDigest TauI 69 TauI 150 

TfiI GAWTC FastDigest TfiI 69 PfeI 135 TfiI 

TliI CTCGAG FastDigest XhoI 71 XhoI 154 PaeR7I, Sfr274I, SlaI, StrI 

Tru1I TTAA 



49 

69 

Tru1I 151 MseI, Tru9I 

Tru9I TTAA 



49 

69 

Tru1I 151 MseI 

TscAI FastDigest TspRI 70 TscAI 151 TspRI 

TseI GCWGC ApeKI 

TsoI 

Tsp45I GTSAC FastDigest NmuCI 53 NmuCI 132 

Tsp509I AATT FastDigest Tsp509I 70 TasI 150 Sse9I, Tsp509I, TspEI 

TspDTI 

TspEI AATT FastDigest Tsp509I 70 TasI 150 Sse9I, Tsp509I 

TspGWI 

TspMI CCCGGG [FastDigest SmaI 65 

Cfr9I m , 

[SmaI 

105 

146 

XmaCI, XmaIm TspRI FastDigest TspRI 70 TscAI 151 TspRI 

TstI 6 Tth111I GACNNNGTC FastDigest PsyI 59 PsyI 139 AspI, PflFI 

Van91I CCANNNNNTGG FastDigest PflMI 56 Van91I 152 AccB7I, BasI, PflMI 

Vha464I CTTAAG FastDigest AflII 15 BspTI 101 AflII, BfrI, Bst98I, BstAFI, MspCI 

VneI GTGCAC FastDigest ApaLI 18 Alw44I 86 ApaLI 

VpaK11BI GGWCC FastDigest AvaII 20 Eco47I 110 AvaII, Bme18I, SinI 

VspI ATTAAT FastDigest AseI 19 VspI 152 AseI, PshBI 

XagI CCTNNNNNAGG FastDigest EcoNI 38 XagI 152 BstENI, EcoNI 

XapI RAATTY FastDigest XapI 70 XapI 153 AcsI, ApoI 

XbaI TCTAGA FastDigest XbaI 71 XbaI 153 

 


216 





XceI RCATGY FastDigest NspI 55 XceI 153 BstNSI, NspI 

XcmI CCANNNNNNNNNTGG 

XhoI CTCGAG FastDigest XhoI 71 XhoI 154 PaeR7I, Sfr274I, SlaI, StrI, TliI 

XhoII RGATCY FastDigest PsuI 58 PsuI 139 BstX2I, BstYI, MflIm , 

XmaCI CCCGGG [FastDigest SmaI 65 

Cfr9I , 

[SmaI 

105 

146 

TspMI, XmaI 

XmaI CCCGGG [FastDigest SmaIm 65 

Cfr9I m , 

[SmaIm 105 

146 

TspMIm , XmaCI 

XmaJI CCTAGG FastDigest AvrII 20 XmaJI 154 AspA2I, AvrII, BlnI 

XmiI GTMKAC FastDigest AccI 13 XmiI 154 AccI, FblI 

XmnI GAANNNNTTC FastDigest PdmIm 55 PdmI m 135 Asp700I, MroXI 

XspI CTAG FastDigest BfaI 22 FspBI 118 BfaI, MaeI 

ZraI GACGTC [FastDigest AatIIm 13 [AatIIm 82 

ZrmI AGTACT FastDigest ScaI 142 ScaI 142 AssI, BmcAI 

Zsp2I ATGCAT FastDigest NsiI 54 Mph1103I 128 EcoT22I, NsiI 


Alphabetic List of Recognition Sequences 

Table 1.26. Recognition sequences of restriction enzymes. 

Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

AACGTT FastDigest AclI Psp1406I 

AAGCTT FastDigest HindIII HindIII 

5 FalI 

AATATT FastDigest SspI SspI 

AATT FastDigest Tsp509I TasI 

ACATGT PscI 

ACCGGT FastDigest AgeI BshTI 

* FastDigest BspMI BveI 

ACCWGGT FastDigest SexAI SexAI 

ACGCGT FastDigest MluI MluI 

* TspGWI 

* BceAI 

ACGT FastDigest TaiI TaiI 

ACGT MaeII 

ACNGT FastDigest TaaI TaaI 

4* BaeI 

5* BsaXI 

ACRYGT AflIII 

ACTAGT FastDigest SpeI BcuI 

* FastDigest BseNI BseNI 

AGATCT FastDigest BglII BglII 

AGCGCT FastDigest AfeI Eco47III 

AGCT FastDigest AluI AluI 

AGGCCT FastDigest StuI Eco147I 

AGTACT FastDigest ScaI ScaI 

ASST SetI 

ATCGAT FastDigest ClaI Bsu15I 

* TspDTI 

ATGCAT FastDigest NsiI Mph1103I 

ATTAAT FastDigest AseI VspI 

ATTTAAAT 

C 

FastDigest SwaI SmiI 

5* CspCI 

CAATTG FastDigest MfeI MunI 

* TaqII 

* AarI 

* BauI 

* AjiI 

CACGTG FastDigest PmlI Eco72I 

CACNNNGTG FastDigest DraIII AdeI 

CACNNNNGTG FastDigest AleI OliI 

BtsI 

* EcoP15I 

CAGCTG FastDigest PvuII PvuII 

CAGNNNCTG FastDigest AlwNI CaiI 

FastDigest TspRI TscAI 

CATATG FastDigest NdeI NdeI 

FastDigest FokI FokI 

FastDigest BseGI BseGI 

BtgZI 

CATG FastDigest NlaIII Hin1II 

CATG FatI 

CATG CviAII 

FastDigest BsrDI BseMI 

CAYNNNNRTG FastDigest MslI RseI 

7* FastDigest AjuI AjuI 

5* CspCI 

FastDigest BseNI BseNI 

CCANNNNNNNNNTGG XcmI 

Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

CCANNNNNNTGG FastDigest BstXI BstXI 

CCANNNNNTGG FastDigest PflMI Van91I 

* BccI 

CCATGG FastDigest NcoI NcoI 

* BseYI 

* FastDigest PspFI GsaI 

* FauI 

CCCGGG Cfr9I 

CCCGGG FastDigest SmaI SmaI 

CCCWGGG PasI 

* FastDigest AciI SsiI 

CCGCGG Cfr42I 

* FastDigest BsrBI MbiI 

CCGG 



HpaII 

MspI 

CCNGG StyD4I 

CCNGG FastDigest ScrFI Bme1390I 

CCNNGG FastDigest BsaJI BseDI 

CCNNNNNNNGG FastDigest BslI BseLI 

CCRYGG BtgI 

CCSGG FastDigest NciI BcnI 

CCTAGG FastDigest AvrII XmaJI 

* FastDigest MnlI MnlI 

* BbvCI 

CCTCGAGG AbsI 

CCTGCAGG FastDigest SbfI SdaI 

* FastDigest Bpu10I Bpu10I 

CCTNAGG FastDigest Bsu36I Eco81I 

CCTNNNNNAGG FastDigest EcoNI XagI 

* HpyAV 

CCWGG EcoRII 

CCWGG FastDigest MvaI MvaI 

CCWWGG FastDigest StyI Eco130I 

6* BcgI 

CGATCG FastDigest PvuI PvuI 

CGCCGGCG FastDigest MreI MreI 

CGCG FastDigest Bsh1236I Bsh1236I 

CGCGCGCG FastDigest MauBI MauBI 

CGGCCG FastDigest EagI Eco52I 

CGGWCCG FastDigest RsrII CpoI 

CGRYCG FastDigest BsiEI Bsh1285I 

CGTACG FastDigest BsiWI Pfl23II 

CGTCGACG SgrDI 

* FastDigest BsmBI Esp3I 

CGWCG Hpy99I 

CMGCKG MspA1I 

SgeI 

6* ArsI 

CRCCGGYG SgrAI 

CTAG FastDigest BfaI FspBI 

BpuEI 

* BspCNI 

* FastDigest BspCNI BseMII 

FastDigest BpmI GsuI 

BseRI 

CTCGAG FastDigest XhoI XhoI 

NmeAIII 

* BauI 

* FastDigest EarI Eam1104I 

 


218 


Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

* FastDigest AcuI Eco57I 

BspCNI 

FastDigest BspCNI BseMII 

BsgI 

CTGCAG FastDigest PstI PstI 

EcoP15I 

* FastDigest BpmI GsuI 

CTNAG FastDigest DdeI HpyF3I 

CTRYAG FastDigest SfcI BfmI 

CTTAAG FastDigest AflII BspTI 

FastDigest AcuI Eco57I 

* BpuEI 

CTYRAG SmoI 

CYCGRG FastDigest AvaI Eco88I 

CYCGRG 

G 

BmeT110I 

6* PsrI 

6* AloI 

* FastDigest MboII MboII 

* FastDigest BbsI BpiI 

FastDigest EarI Eam1104I 

FastDigest SapI LguI 

HpyAV 

6* BarI 


GAANNNNTTC FastDigest PdmI PdmI 

* FastDigest Mva1269I Mva1269I 

GAATTC FastDigest EcoRI EcoRI 

* TaqII 

* FastDigest HgaI CseI 

GACGTC ZraI 

GACGTC FastDigest AatII AatII 

* AjiI 

GACNNNGTC FastDigest PsyI PsyI 

GACNNNNGTC FastDigest PshAI BoxI 

GACNNNNNGTC FastDigest Eam1105I Eam1105I 

GACNNNNNNGTC FastDigest DrdI DrdI 

6* ArsI 

FastDigest MlyI SchI 

PleI 

FastDigest Alw26I Alw26I 

FastDigest Eco31I Eco31I 

FastDigest BsmBI Esp3I 


GAGCTC FastDigest SacI SacI 

GAGCTC FastDigest Ecl136II Ecl136II 

FastDigest MnlI MnlI 

* BseRI 

5 FastDigest BplI BplI 

* FastDigest MlyI SchI 

* PleI 

GANTC FastDigest HinfI HinfI 

GATATC FastDigest EcoRV Eco32I 

GATC 



Bsp143I 

MboI 

Gm6ATC FastDigest DpnI DpnI 

GATC BstKTI 

BspPI 

FastDigest SfaNI LweI 

BccI 

GATNNNNATC FastDigest BsaBI BseJI 


Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

GAWTC FastDigest TfiI PfeI 

* FastDigest BsrDI BseMI 

* 



Lsp1109I 

BseXI 

FastDigest BspMI BveI 

AarI 

* BtsI 

6* BcgI 

6 AlfI 

GCANNNNNTGC BstAPI 

* FastDigest SfaNI LweI 

GCATGC FastDigest SphI PaeI 

FastDigest Mva1269I Mva1269I 

GCCCGGGC SrfI 

* NmeAIII 

GCCGGC NgoMIV 

GCCGGC FastDigest NaeI PdiI 

BceAI 

GCCNNNNNGGC FastDigest BglI BglI 

GCGATCGC FastDigest AsiSI SfaAI 

* BtgZI 

GCGC FastDigest HinP1I Hin6I 

GCGC FastDigest HhaI HhaI 

GCCG GlaI 

GCGCGC FastDigest BssHII PauI 


GCGGCCGC FastDigest NotI NotI 

FauI 

FastDigest HgaI CseI 

GCNGC FastDigest Fnu4HI SatI 

GCNGC BisI 

GCNGC BlsI 

GCNNGC Cac8I 

GCNNNNNNNGC FastDigest HpyF10VI HpyF10VI 

GCSGC FastDigest TauI TauI 

GCTAGC FastDigest BmtI BspOI 

GCTAGC FastDigest NheI NheI 

* FastDigest SapI LguI 

* BbvCI 

 



Lsp1109I 

BseXI 

* BseYI 


GCTNAGC FastDigest BlpI Bpu1102I 


GCWGC TseI 

GDGCHC FastDigest Bsp1286I SduI 

5* BsaXI 

6* AloI 

BfuI 

* BspPI 

GGATCC FastDigest BamHI BamHI 

* FastDigest FokI FokI 

* FastDigest BseGI BseGI 

GGCC FastDigest HaeIII BsuRI 

GGCCGGCC FseI 

GGCCNNNNNGGCC FastDigest SfiI SfiI 

GGCGCC FastDigest EheI EheI 

GGCGCC NarI 

GGCGCC SspDI 

GGCGCC BbeI


Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

GGCGCGCC FastDigest AscI SgsI 

* EciI 

* FastDigest BsmFI FaqI 

GGGCCC FastDigest Bsp120I Bsp120I 

GGGCCC FastDigest ApaI ApaI 

GGGWCCC FastDigest SanDI SanDI 

GGNCC FastDigest Sau96I Cfr13I 

GGNCC BmgT120I 

GGNNCC FastDigest NlaIV BspLI 

GGTACC FastDigest KpnI KpnI 

GGTACC FastDigest Acc65I Acc65I 

* FastDigest Eco31I Eco31I 

* HphI 

GGTNACC FastDigest Eco91I Eco91I 

GGWCC FastDigest AvaII Eco47I 

GGYRCC FastDigest BanI BshNI 

GKGCMC FastDigest Bme1580I BseSI 

GRCGYC FastDigest BsaHI Hin1I 

GRGCYC Eco24I 

4* BaeI 

GTAC FastDigest RsaI RsaI 

GTAC FastDigest Csp6I Csp6I 

6* BarI 

6* PsrI 

GTATAC FastDigest BstZ17I Bst1107I 

* BfuI 

FastDigest BsmFI FaqI 

GTCGAC FastDigest SalI SalI 

* FastDigest Alw26I Alw26I 

FastDigest BbsI BpiI 

GTGCAC FastDigest ApaLI Alw44I 

* BsgI 

GTMKAC FastDigest AccI XmiI 

GTNAC MaeIII 

GTNNAC FastDigest Hpy8I Hpy8I 

GTSAC FastDigest NmuCI NmuCI 

GTTAAC FastDigest HpaI KspAI 

GTTTAAAC FastDigest MssI MssI 

MmeI 

GTYRAC FastDigest HincII HincII 

GWGCWC 

R 


RAATTY FastDigest XapI XapI 

RCATGY FastDigest NspI XceI 

RCCGGY FastDigest BsrFI Cfr10I 

RGATCY FastDigest PsuI PsuI 

RGCGCY FastDigest HaeII HaeII 

RGCY 

RGGNCCY FastDigest EcoO109I EcoO109I 

RGGWCCY FastDigest PpuMI Psp5II 

RTGCGCAY 

T 

FastDigest FspAI FspAI 

TACGTA FastDigest SnaBI Eco105I 

HphI 

TCATGA FastDigest BspHI PagI 

EciI 

TCCGGA FastDigest Kpn2I Kpn2I 

TspGWI 

TaqII 

TCCNGGA FastDigest PfoI PfoI 

* MmeI 

Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

TCGA FastDigest TaqI TaqI 

TCGCGA FastDigest NruI Bsp68I 

TCNGA Hpy188I 

TCNNGA Hpy188III 

TCTAGA FastDigest XbaI XbaI 

FastDigest MboII MboII 

TGATCA FastDigest BclI BclI 

TGCA HpyCH4V 

TGCGCA FastDigest FspI NsbI 

TGGCCA FastDigest MscI MlsI 

TaqII 


TGTACA FastDigest Bsp1407I Bsp1407I 

TTAA 



Tru1I 

TTAATTAA FastDigest PacI PacI 

TTATAA FastDigest PsiI AanI 

TspDTI 

TTCGAA FastDigest Bsp119I Bsp119I 

TTSAA AgsI 

TTTAAA 

V 

FastDigest DraI DraI 

VCTCGAGB 

W 

PspXI 

WCCGGW BsaWI 

WGTACW 

Y 

FastDigest TatI TatI 

YACGTR FastDigest BsaAI Ppu21I 

YATR FaiI 

YGGCCR EaeI 

Note 

* Asymetric sequences. 







M = A or C; V = A, C or G; 


220 

Enzyme Recognizing 2 bp Long Targets 

Table 1.27. Enzyme recognizing 2 bp long targets. 

Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

SgeI 

Alphabetic List of Enzymes 

Recognizing 4 bp Long Targets 

Table 1.28. Enzymes recognizing 4 bp long targets. 

Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

AATT FastDigest Tsp509I TasI 

ACGT FastDigest TaiI TaiI 

ACGT MaeII 

ACNGT FastDigest TaaI TaaI 


ASST SetI 

CATG FastDigest NlaIII Hin1II 

CATG FatI 

CATG CviAII 


CCGG 



HpaII 

MspI 

CCNGG StyD4I 

CCNGG FastDigest ScrFI Bme1390I 

CCNNGG FastDigest BsaJI BseDI 

CCNNNNNNNGG FastDigest BslI BseLI 

* FastDigest MnlI MnlI 


CTAG FastDigest BfaI FspBI 

CTNAG FastDigest DdeI HpyF3I 

FastDigest MnlI MnlI 

GANTC FastDigest HinfI HinfI 

GATC 



MboI 

Bsp143I 

Gm6ATC FastDigest DpnI DpnI 

GATC BstKTI 

GCGC FastDigest HinP1I Hin6I 

GCGC FastDigest HhaI HhaI 

GCGC GlaI 


GCNGC FastDigest Fnu4HI SatI 

GCNGC BisI 

GCNGC BlsI 

GCNNGC Cac8I 

GCNNNNNNNGC FastDigest HpyF10VI HpyF10VI 


GGNCC FastDigest Sau96I Cfr13I 

GGNCC BmgT120I 



GTAC FastDigest Csp6I Csp6I 

GTNAC MaeIII 


RGCY 

TCGA FastDigest TaqI TaqI 

TCNGA Hpy188I 

TCNNGA Hpy188III 

TGCA HpyCH4V 

TTAA 



Tru1I 

YATR FaiI 

Note 








Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

* TspGWI 

* BceAI 

* FastDigest BseNI BseNI 

* TspDTI 

FastDigest TspRI TscAI 

FastDigest FokI FokI 

FastDigest BseGI BseGI 

FastDigest BseNI BseNI 

* BccI 

* FauI 

CCSGG FastDigest NciI BcnI 

* HpyAV 

CCWGG EcoRII 

CCWGG FastDigest MvaI MvaI 

CGWCG Hpy99I 

* BspCNI 

* FastDigest BspCNI BseMII 

BspCNI 

FastDigest BspCNI BseMII 

* FastDigest MboII MboII 

HpyAV 

* FastDigest HgaI CseI 


PleI 



* PleI 

BspPI 

FastDigest SfaNI LweI 

BccI 

GAWTC FastDigest TfiI PfeI 

* 



Lsp1109I 

BseXI 

* FastDigest SfaNI LweI 

BceAI 

FauI 

FastDigest HgaI CseI 

GCSGC FastDigest TauI TauI 

 



Lsp1109I 

BseXI 

GCWGC TseI 

* BspPI 

* FastDigest FokI FokI 

* FastDigest BseGI BseGI 

* FastDigest BsmFI FaqI 

* HphI 

GGWCC FastDigest AvaII Eco47I 

FastDigest BsmFI FaqI 

* FastDigest Alw26I Alw26I 

GTSAC FastDigest NmuCI NmuCI 

HphI 

TspGWI 

FastDigest MboII MboII 

TspDTI 

TTSAA AgsI 






Alphabetic List of Enzymes Recognizing 6 bp Long Targets 


Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

AACGTT FastDigest AclI Psp1406I 

AAGCTT FastDigest HindIII HindIII 

5 FalI 


ACATGT PscI 

ACCGGT FastDigest AgeI BshTI 

* FastDigest BspMI BveI 

ACGCGT FastDigest MluI MluI 

5* BsaXI 

ACRYGT AflIII 

ACTAGT FastDigest SpeI BcuI 

AGATCT FastDigest BglII BglII 




ATCGAT FastDigest ClaI Bsu15I 

ATGCAT FastDigest NsiI Mph1103I 

ATTAAT FastDigest AseI VspI 

CAATTG FastDigest MfeI MunI 

* TaqII 

* BauI 

* AjiI 


CACNNNGTG FastDigest DraIII AdeI 


BtsI 

* EcoP15I 


CAGNNNCTG FastDigest AlwNI CaiI 

CATATG FastDigest NdeI NdeI 

BtgZI 

FastDigest BsrDI BseMI 


CCANNNNNNNNNTGG XcmI 

CCANNNNNNTGG FastDigest BstXI BstXI 

CCANNNNNTGG FastDigest PflMI Van91I 

CCATGG FastDigest NcoI NcoI 

* BseYI 


CCCGGG Cfr9I 


CCGCGG Cfr42I 


CCRYGG BtgI 

CCTAGG FastDigest AvrII XmaJI 


CCTNAGG FastDigest Bsu36I Eco81I 

CCTNNNNNAGG FastDigest EcoNI XagI 

CCWWGG FastDigest StyI Eco130I 

6* BcgI 

CGATCG FastDigest PvuI PvuI 

CGGCCG FastDigest EagI Eco52I 

CGRYCG FastDigest BsiEI Bsh1285I 

CGTACG FastDigest BsiWI Pfl23II 

* FastDigest BsmBI Esp3I 

CMGCKG MspA1I 

BpuEI 

FastDigest BpmI GsuI 

BseRI 

CTCGAG FastDigest XhoI XhoI 

NmeAIII 

* BauI 

* FastDigest EarI Eam1104I 

* FastDigest AcuI Eco57I 

Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

BsgI 

CTGCAG FastDigest PstI PstI 

EcoP15I 

* FastDigest BpmI GsuI 

CTRYAG FastDigest SfcI BfmI 

CTTAAG FastDigest AflII BspTI 

FastDigest AcuI Eco57I 

* BpuEI 

CTYRAG SmoI 

CYCGRG FastDigest AvaI Eco88I 

CYCGRG BmeT110I 

* FastDigest BbsI BpiI 

FastDigest EarI Eam1104I 


* FastDigest Mva1269I Mva1269I 

GAATTC FastDigest EcoRI EcoRI 

* TaqII 

GACGTC ZraI 

GACGTC FastDigest AatII AatII 

* AjiI 

GACNNNGTC FastDigest PsyI PsyI 


GACNNNNNGTC FastDigest Eam1105I Eam1105I 

GACNNNNNNGTC FastDigest DrdI DrdI 

FastDigest Eco31I Eco31I 

FastDigest BsmBI Esp3I 


GAGCTC FastDigest SacI SacI 


* BseRI 




* FastDigest BsrDI BseMI 

FastDigest BspMI BveI 

* BtsI 

6* BcgI 

6 AlfI 

GCANNNNNTGC BstAPI 

GCATGC FastDigest SphI PaeI 

FastDigest Mva1269I Mva1269I 

* NmeAIII 

GCCGGC NgoMIV 


GCCNNNNNGGC FastDigest BglI BglI 

* BtgZI 

GCGCGC FastDigest BssHII PauI 

GCTAGC FastDigest BmtI BspOI 

GCTAGC FastDigest NheI NheI 

* BseYI 


GCTNAGC FastDigest BlpI Bpu1102I 


GDGCHC FastDigest Bsp1286I SduI 

5* BsaXI 

BfuI 

GGATCC FastDigest BamHI BamHI 


GGCGCC NarI 

GGCGCC SspDI 

GGCGCC BbeI 

* EciI 

GGGCCC FastDigest Bsp120I Bsp120I 

GGGCCC FastDigest ApaI ApaI 

 


222 


Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

GGTACC FastDigest KpnI KpnI 

GGTACC FastDigest Acc65I Acc65I 

* FastDigest Eco31I Eco31I 

GGTNACC FastDigest Eco91I Eco91I 

GGYRCC FastDigest BanI BshNI 

GKGCMC FastDigest Bme1580I BseSI 

GRCGYC FastDigest BsaHI Hin1I 

GRGCYC Eco24I 


* BfuI 

GTCGAC FastDigest SalI SalI 

FastDigest BbsI BpiI 

GTGCAC FastDigest ApaLI Alw44I 

* BsgI 

GTMKAC FastDigest AccI XmiI 


MmeI 


GWGCWC FastDigest Alw21I Alw21I 

RAATTY FastDigest XapI XapI 

RCATGY FastDigest NspI XceI 

RCCGGY FastDigest BsrFI Cfr10I 

RGATCY FastDigest PsuI PsuI 

RGCGCY FastDigest HaeII HaeII 

RGGNCCY FastDigest EcoO109I EcoO109I 


TCATGA FastDigest BspHI PagI 

EciI 

TCCGGA FastDigest Kpn2I Kpn2I 

TCCNGGA FastDigest PfoI PfoI 

* MmeI 


TaqII 

TCTAGA FastDigest XbaI XbaI 

TGATCA FastDigest BclI BclI 



TaqII 

TGTACA FastDigest Bsp1407I Bsp1407I 


TTCGAA FastDigest Bsp119I Bsp119I 

TTTAAA FastDigest DraI DraI 

WCCGGW BsaWI 

WGTACW FastDigest TatI TatI 


YGGCCR EaeI 

Note 








Specificity 

FastDigest 

Conventional 

5’3’ 

enzyme 

enzyme 

ACCWGGT FastDigest SexAI SexAI 

4* BaeI 

5* CspCI 

* AarI 


5* CspCI 

CCCWGGG PasI 

* BbvCI 

CGGWCCG FastDigest RsrII CpoI 

6* ArsI 

6* PsrI 

6* AloI 

FastDigest SapI LguI 

6* BarI 


6* ArsI 

AarI 

* FastDigest SapI LguI 

* BbvCI 

6* AloI 

GGGWCCC FastDigest SanDI SanDI 

* BaeI 

6* BarI 

6* PsrI 

RGGWCCY FastDigest PpuMI Psp5II 




Specificity 5’3’ 

FastDigest 

enzyme 

Conventional 

enzyme 

ATTTAAAT FastDigest SwaI SmiI 

CCTCGAGG AbsI 

CCTGCAGG FastDigest SbfI SdaI 

CGCCGGCG FastDigest MreI MreI 

CGCGCGCG FastDigest MauBI MauBI 

CGTCGACG SgrDI 

CRCCGGYG SgrAI 

GCCCGGGC SrfI 

GCGATCGC FastDigest AsiSI SfaAI 

GCGGCCGC FastDigest NotI NotI 

GGCCGGCC FseI 

GGCCNNNNNGGCC FastDigest SfiI SfiI 

GGCGCGCC FastDigest AscI SgsI 


RTGCGCAY FastDigest FspAI FspAI 

TTAATTAA FastDigest PacI PacI 

VCTCGAGB PspXI 


R = G or A; K = G or T; B = C, G or T; M = A or C; 

Y = C or T; S = C or G; D = A, G or T; V = A, C or G. 

W = A or T; H = A, C or T; N = G, A, T or C;

Commercial Restriction Enzymes Generating 5’-protruding Ends 

Table 1.33. Commercial restriction enzymes generating 5’-protruding ends. 

Recognition FastDigest 

Conventional 

5’-protruding end (5’ 3’) 

sequence 

enzyme 

enzyme 

1 nt 2 nt 3 nt 4 nt 5 nt 

AACGTT FastDigest AclI Psp1406I CG 

AAGCTT FastDigest HindIII HindIII AGCT 

AATT FastDigest Tsp509I TasI AATT 

ACATGT PscI CATG 

ACCGGT FastDigest AgeI BshTI CCGG 

* FastDigest BspMI BveI NNNN 

ACCWGGT FastDigest SexAI SexAI CCWGG 

ACGCGT FastDigest MluI MluI CGCG 

* BceAI NN 

ACGT MaeII CG 

ACRYGT AflIII CRYG 

ACTAGT FastDigest SpeI BcuI CTAG 

AGATCT FastDigest BglII BglII GATC 

ATCGAT FastDigest ClaI Bsu15I CG 

ATTAAT FastDigest AseI VspI TA 

CAATTG FastDigest MfeI MunI AATT 

* AarI NNNN 

* BauI ACGA 

* EcoP15I NN 

CATATG FastDigest NdeI NdeI TA 

FastDigest FokI FokI NNNN 

BtgZI NNNN 

CATG FatI CATG 

CATG CviAII AT 

* BccI N 

CCATGG FastDigest NcoI NcoI CATG 

* BseYI CCAG 

* FauI NN 

CCCGGG Cfr9I CCGG 

CCCWGGG PasI CWG 

* FastDigest AciI SsiI CG 

CCGG 



HpaII 

MspI 

CG 

CCNGG StyD4I CCNGG 

CCNGG FastDigest ScrFI Bme1390I N 

CCNNGG FastDigest BsaJI BseDI CNNG 

CCRYGG BtgI CRYG 

CCSGG FastDigest NciI BcnI S 

CCTAGG FastDigest AvrII XmaJI CTAG 

* BbvCI TCA 

CCTCGAGG AbsI TCGA 

* FastDigest Bpu10I Bpu10I TNA 

CCTNAGG FastDigest Bsu36I Eco81I TNA 

CCTNNNNNAGG FastDigest EcoNI XagI N 

CCWGG EcoRII CCWGG 

CCWGG FastDigest MvaI MvaI W 

CCWWGG FastDigest StyI Eco130I CWWG 

CGCCGGCG FastDigest MreI MreI CCGG 

CGCGCGCG FastDigest MauBI MauBI CGCG 

CGGCCG FastDigest EagI Eco52I GGCC 

CGGWCCG FastDigest RsrII CpoI GWC 

CGTACG FastDigest BsiWI Pfl23II GTAC 

SgeI NNNN 

CGTCGACG SgrDI TCGA 

* FastDigest BsmBI Esp3I NNNN 

CRCCGGYG SgrAI CCGG 

 


224 



Conventional 


sequence 

enzyme 

enzyme 


CTAG FastDigest BfaI FspBI TA 

CTCGAG FastDigest XhoI XhoI TCGA 

* BauI TCGT 

* FastDigest EarI Eam1104I NNN 

EcoP15I NN 

CTNAG FastDigest DdeI HpyF3I TNA 

CTRYAG FastDigest SfcI BfmI TRYA 

CTTAAG FastDigest AflII BspTI TTAA 

CTYRAG SmoI TYRA 

CYCGRG FastDigest AvaI Eco88I YCGR 

CYCGRG BmeT110I CG 

* FastDigest BbsI BpiI NNNN 

FastDigest EarI Eam1104I NNN 

FastDigest SapI LguI NNN 

GAATTC FastDigest EcoRI EcoRI AATT 

* FastDigest HgaI CseI NNNNN 

GACNNNGTC FastDigest PsyI PsyI N 

PleI N 

FastDigest Alw26I Alw26I NNNN 

FastDigest Eco31I Eco31I NNNN 

FastDigest BsmBI Esp3I NNNN 

* PleI N 

GANTC FastDigest HinfI HinfI ANT 

GATC 



MboI 

Bsp143I 

GATC 

BspPI N 

FastDigest SfaNI LweI NNNN 

BccI N 

GAWTC FastDigest TfiI PfeI AWT 

* 


FastDigest BseXI 

Lsp1109I 

BseXI 

NNNN 

FastDigest BspMI BveI NNNN 

AarI NNNN 

* FastDigest SfaNI LweI NNNN 

GCCGGC NgoMIV CCGG 

BceAI NN 

* BtgZI NNNN 

GCGC FastDigest HinP1I Hin6I CG 

GCGCGC FastDigest BssHII PauI CGCG 

* FastDigest AciI SsiI CG 

GCGGCCGC FastDigest NotI NotI GGCC 

FauI NN 

FastDigest HgaI CseI NNNNN 

GCNGC FastDigest Fnu4HI SatI N 

GCTAGC FastDigest NheI NheI CTAG 

* FastDigest SapI LguI NNN 

* BbvCI TGA 

 


FastDigest BseXI 

Lsp1109I 

BseXI 

NNNN 

* BseYI CTGG 

GCTNAGC FastDigest BlpI Bpu1102I TNA 

* FastDigest Bpu10I Bpu10I TNA 

GCWGC TseI CWG 

* BspPI N 

GGATCC FastDigest BamHI BamHI GATC 

* FastDigest FokI FokI NNNN 

GGCGCC NarI CG 

GGCGCC SspDI GCGC 

GGCGCGCC FastDigest AscI SgsI CGCG 

* FastDigest BsmFI FaqI NNNN 

 




Conventional 


sequence 

enzyme 

enzyme 


GGGCCC FastDigest Bsp120I Bsp120I GGCC 

GGGWCCC FastDigest SanDI SanDI GWC 

GGNCC FastDigest Sau96I Cfr13I GNC 

GGNCC BmgT120I N 

GGTACC FastDigest Acc65I Acc65I GTAC 

* FastDigest Eco31I Eco31I NNNN 

GGTNACC FastDigest Eco91I Eco91I GTNAC 

GGWCC FastDigest AvaII Eco47I GWC 

GGYRCC FastDigest BanI BshNI GYRC 

GRCGYC FastDigest BsaHI Hin1I CG 

GTAC FastDigest Csp6I Csp6I TA 

FastDigest BsmFI FaqI NNNN 

GTCGAC FastDigest SalI SalI TCGA 

* FastDigest Alw26I Alw26I NNNN 

FastDigest BbsI BpiI NNNN 

GTGCAC FastDigest ApaLI Alw44I TGCA 

GTMKAC FastDigest AccI XmiI MK 

GTNAC MaeIII GTNAC 

GTSAC FastDigest NmuCI NmuCI GTSAC 

RAATTY FastDigest XapI XapI AATT 

RCCGGY FastDigest BsrFI Cfr10I CCGG 

RGATCY FastDigest PsuI PsuI GATC 

RGGNCCY FastDigest EcoO109I EcoO109I GNC 

RGGWCCY FastDigest PpuMI Psp5II GWC 

TCATGA FastDigest BspHI PagI CATG 

TCCGGA FastDigest Kpn2I Kpn2I CCGG 

TCCNGGA FastDigest PfoI PfoI CCNGG 

TCGA FastDigest TaqI TaqI CG 

TCNNGA Hpy188III NN 

TCTAGA FastDigest XbaI XbaI CTAG 

TGATCA FastDigest BclI BclI GATC 

TGTACA FastDigest Bsp1407I Bsp1407I GTAC 

TTAA 



Tru1I TA 

TTCGAA FastDigest Bsp119I Bsp119I CG 

VCTCGAGB PspXI TCGA 

WCCGGW BsaWI CCGG 

WGTACW FastDigest TatI TatI GTAC 

YGGCCR EaeI GGCC 

Note 










226 

Commercial Restriction Enzymes Generating 3’-protruding Ends 


Recognition sequence 

 

FastDigest 

enzyme 

Conventional 

enzyme 

1 nt 



* TspGWI NN 

ACGT FastDigest TaiI TaiI ACGT 

ACNGT FastDigest TaaI TaaI N 

* FastDigest BseNI BseNI GN 

ASST SetI ASST 

* TspDTI NN 

ATGCAT FastDigest NsiI Mph1103I TGCA 

* TaqII NN 

CACNNNGTG FastDigest DraIII AdeI NNN 

BtsI NN 

CAGNNNCTG FastDigest AlwNI CaiI NNN 

FastDigest TspRI TscAI NNCASTGNN 

FastDigest BseGI BseGI NN 

CATG FastDigest NlaIII Hin1II CATG 

FastDigest BsrDI BseMI NN 

FastDigest BseNI BseNI NC 

CCANNNNNNNNNTGG XcmI N 

CCANNNNNNTGG FastDigest BstXI BstXI NNNN 

CCANNNNNTGG FastDigest PflMI Van91I NNN 

* FastDigest PspFI GsaI CCAG 

CCGCGG Cfr42I GC 

CCNNNNNNNGG FastDigest BslI BseLI NNN 

* FastDigest MnlI MnlI N 

CCTGCAGG FastDigest SbfI SdaI TGCA 

* HpyAV N 

CGATCG FastDigest PvuI PvuI AT 

CGRYCG FastDigest BsiEI Bsh1285I RY 

CGWCG Hpy99I CGWCG 

BpuEI NN 

* BspCNI NN 

* FastDigest BspCNI BseMII NN 

FastDigest BpmI GsuI NN 

BseRI NN 

NmeAIII NN 

* FastDigest AcuI Eco57I NN 

BspCNI NN 

FastDigest BspCNI BseMII NN 

BsgI NN 

CTGCAG FastDigest PstI PstI TGCA 

* FastDigest BpmI GsuI NN 

FastDigest AcuI Eco57I NN 

* BpuEI NN 

* FastDigest MboII MboII N 

HpyAV N 

* FastDigest Mva1269I Mva1269I CN 

* TaqII NN 

GACGTC FastDigest AatII AatII ACGT 

GACNNNNNGTC FastDigest Eam1105I Eam1105I N 

GACNNNNNNGTC FastDigest DrdI DrdI NN 

GAGCTC FastDigest SacI SacI AGCT 

FastDigest MnlI MnlI N 

* BseRI NN 

GATC BstKTI AT 

* FastDigest BsrDI BseMI NN 

* BtsI NN 

GCANNNNNTGC BstAPI NNN 

GCATGC FastDigest SphI PaeI CATG 

FastDigest Mva1269I Mva1269I NG 

* NmeAIII NN 



Recognition sequence FastDigest 

enzyme 

Conventional 

enzyme 

1 nt 



GCCNNNNNGGC FastDigest BglI BglI NNN 

GCGATCGC FastDigest AsiSI SfaAI AT 

GCGC FastDigest HhaI HhaI CG 

GCNNNNNNNGC FastDigest HpyF10VI HpyF10VI NNN 

GCSGC FastDigest TauI TauI CSG 

GCTAGC FastDigest BmtI BspOI CTAG 

* FastDigest PspFI GsaI CTGG 

GDGCHC FastDigest Bsp1286I SduI DGCH 

BfuI N 

* FastDigest BseGI BseGI NN 

GGCCGGCC FseI CCGG 

GGCCNNNNNGGCC FastDigest SfiI SfiI NNN 

GGCGCC BbeI GCGC 

* EciI NN 

GGGCCC FastDigest ApaI ApaI GGCC 

GGTACC FastDigest KpnI KpnI GTAC 

* HphI N 

GKGCMC FastDigest Bme1580I BseSI KGCM 

GRGCYC Eco24I RGCY 

* BfuI N 

* BsgI NN 

MmeI NN 

GWGCWC FastDigest Alw21I Alw21I WGCW 

RCATGY FastDigest NspI XceI CATG 

RGCGCY FastDigest HaeII HaeII GCGC 

HphI N 

EciI NN 

TspGWI NN 

* MmeI NN 

TaqII NN 

TCNGA Hpy188I N 

FastDigest MboII MboII N 

TaqII NN 

TTAATTAA FastDigest PacI PacI AT 

TspDTI NN 

TTSAA AgsI S 

Note 










228 

Commercial Restriction 

Enzymes Generating Blunt Ends 

Table 1.35. Commercial restriction enzymes generating 

blunt ends. 

Reccognition FastDigest 

Conventional 

sequence enzyme 

enzyme 






ATTTAAAT FastDigest SwaI SmiI 

* AjiI 








CMGCKG MspA1I 


GACGTC ZraI 

* AjiI 







GATC FastDigest DpnI DpnI 


GCCCGGGC SrfI 


GCGC GlaI 

GCNNGC Cac8I 










RGCY 

RTGCGCAY FastDigest FspAI FspAI 



TGCA HpyCH4V 




TTTAAA FastDigest DraI DraI 


YATR FaiI 

Note 





Commercial Restriction Enzymes Cleaving DNA on 

the Both Sides of their Recognition Sequence 

Table 1.36. Commercial restriction enzymes cleaving DNA on 

the both sides of their recognition sequence. 

Recognition sequence 

FastDigest 

enzyme 

Conventional 

enzyme 

5 FalI 

5 BsaXI 

4 BaeI 

5 CspCI 

7 FastDigest AjuI AjuI 

5 CspCI 

6 BcgI 

6 ArsI 

7 FastDigest AjuI AjuI 

6 PsrI 

6 AloI 

6 BarI 

6 ArsI 


6 BcgI 

6 AlfI 

6 AloI 

5 BsaXI 

4 BaeI 

6 BarI 

6 PsrI 






Troubleshooting Guide 

1.1 

Inactive 

enzyme 

1.2.1 

Suboptimal 

digestion 

protocol 

1.2.2 

Improper 

enzyme 

dilution 

1.2.3 

Improper 

reaction 

assembly 

1.2.4 

Excess 

glycerol 

1.2.5 

Suboptimal 

DNA 

concentration 

1 

Incomplete 

digestion, no 

digestion 

Assess enzyme 

activity with 

control DNA 

1.2 

Suboptimal 

reaction 

conditions 

Active 

enzyme 

1.3.1 

Contaminants 

in DNA 

solution 

Inhibition 

1.3.4.1 

Supercoiled 

plasmid 

DNA 

Digestion problem 

1.3 

Unsuitable or 

contaminated 

DNA 

Evaluate 

inhibition by 

template DNA 

solution 

1.3.4.2 

Proximity of 

site to DNA 

ends 

1.3.2 

Absence of 

recognition 

sites 

1.3.3 

Cleavage 

blocked by 

methylation 

1.3.4 

Structure of 

DNA 

substrate 

1.4 

Water 

contains 

impurities 

No inhibition 

1.3.4.3 

At least two 

sites required 

2 

Unexpected 

cleavage 

pattern 

2.1 

Star 

activity 

2.2 

Contamination 

with another 

RE 

2.3 

Contamination 

with another 

DNA 

2.4 

Incomplete 

DNA digestion 

2.5 

Gel shift 

2.6 

Unexpected 

sites in 

template DNA 

1.3.4.4 

Site 

preference 

3 

Diffuse DNA 

bands on gel 

3.1 

Gel shift 

3.2 

Contaminated 

reagents 


230 

Table 1.37. Troubleshooting guide for DNA digestion. 

Problem Possible cause and recommended solution 

1. Incomplete digestion or 

no digestion 


Assess enzyme activity 

The restriction enzyme may lose activity due to improper storage or handling. Perform a digestion reaction with 


1.1. Inactive enzyme. 

If the enzyme does not cut the control DNA: 

 

 

 

 

1.2. Suboptimal reaction conditions. 

1.2.1. Suboptimal digestion protocol. 

 

 

DNA, as well as PCR products. Please refer to Table 1.3 on p.73 for specific recommendations. 

enzymes, use FastDigest or FastDigest Green Buffer. All FastDigest enzymes are 100% active 

in these buffers. 

 

For double digestions, follow the recommendations of the DoubleDigest engine at 

www.thermoscientific.com/doubledigest. 

 

 

with enzymes requiring different incubation temperatures, perform sequential DNA cleavage: complete 

the first digestion reaction at the lower temperature, add the second enzyme and increase the digestion 

temperature for the second enzyme cleavage. 

 

increase in salt concentration may reduce enzyme activity. For thermophilic enzymes use a heat block with a 

hot bonnet; e.g., a PCR cycler. 

1.2.2. Improper dilution of conventional enzyme. 

 

enzymes diluted with this buffer are stable for at least 3-4 weeks at -20°C. 

 

 

enzymes, which are experimentally tested on four different 

see 

1.2.3. Improper reaction assembly. 

 

 

1.2.4. Excess glycerol in the reaction mixture. 

 

enzyme added to the mixture should not exceed 1/10 of the total reaction volume. 

 

Hin6I, HinfI, Mph1103I, Mva1269I and NcoI. 

1.2.5. Suboptimal DNA concentration. 

The optimal range of DNA concentration in the reaction mixture is 0.02-0.1 μg/μl. 

1.3. Unsuitable DNA template or contaminated DNA solution. 

If the enzyme is active in the control digest, assay the substrate DNA solution for inhibitory contaminants in 


digest with two templates, control and sample, in one reaction mixture. Do not exceed the optimal DNA 

 

see 

 

see 

see see 

Note 

Always ensure that the control DNA contains a recognition site for the enzyme present in the reaction. For example, there is no NotI 

recognition site in lambda DNA.




no digestion 

1.3.1. Contaminants in the DNA solution. 

 

 

see 260/280 ratio should be 1.8-2.0. To remove EDTA and salts, wash the pellet 

with 70% cold ethanol. 

 

 

enzyme buffer see protocols on p.72 or p.158. 

 

centrifugation for 10 min at 10,000 rpm and ensure that no resin is carried over while transferring the DNA 

solution into a new tube. 

1.3.2. The substrate DNA does not contain a recognition sequence for the restriction enzyme. 

 

 

see 

see 

 

recognition site. 

1.3.3. Methylation effects. 

 

 

impairs or blocks DNA digestion with the enzyme. See Digestion of Methylated DNA on p.173 and use 

Table 1.13 and Table 1.20 on pp.174-179. 

If methylation impairs or blocks DNA cleavage: 

E.coli dam – , dcm – E.coli GM2163 dam – , dcm – 

engine at www.thermoscientific.com/research or check the Thermo Scientific 

molecular biology tools catalog for the availability of a restriction enzyme isoschizomer not sensitive 

to DNA methylation. 

 

of unmethylated DNA will result in no DNA cleavage. Propagate your plasmid in E.coli dam + or dcm + strains 

E.coli 

DpnI or SgeI recognition sequences. Alternatively, in the case of DpnI, the neoschizomers Bsp143I or MboI 

can be used to digest non-methylated DpnI recognition sites. 

Note 

When PCR is carried out with standard dNTPs and non-methylated primers the resulting DNA product is not methylated. 

1.3.4. Structure of substrate DNA. 

1.3.4.1. Supercoiled plasmid DNA. 

enzymes which are qualified for supercoiled DNA and provided with specific 

see 

 

 

of the enzyme or refer to the Certificate of Analysis. 

1.3.4.2. Proximity of the recognition sequence to the DNA ends. 

Some restriction enzymes cleave DNA poorly if the recognition site is too close to the end of the DNA molecule. 

enzymes refer to Table 1.3 on p.73 or the product description to determine the 

effectiveness of restriction enzyme cleavage at the ends of DNA. 

 

1.3.4.3. Restriction enzyme requires at least two sites per DNA molecule for optimal activity. 

Some restriction enzymes such as AarI, BveI, Cfr10I, Cfr42I, Eco57I, EcoRII, LweI, SfiI require at least two 

see Site Preferences by Restriction 

 

containing the recognition site. 

1.3.4.4. Site Preferences by Restriction Enzymes. 

The DNA sequence surrounding the recognition site may influence the efficiency of digestion. Some DNA sites are 

see 

 

see Table 1.25 on p.205 or REsearch engine at www.thermoscientific.com/research 

 


232 




no digestion 

2. Unexpected cleavage pattern 


1.4. Water contains impurities. 

 

 

18 M. 

 

see 

2.1. Star activity (relaxed specificity) of restriction enzyme see 

restriction enzymes. For these enzymes, incubation time without star activity has been 

experimentally determined. 

 

 

 

enzymes, refer to Table 1.3 on p.73 for maximum incubation 

times. 

 

resulting increase in glycerol concentration may cause star activity. 

2.2. Contamination with another restriction enzyme. 

The restriction enzyme or buffer may be contaminated with another restriction enzyme due to improper 

handling. Use a new tube of enzyme and/or buffer. 

2.3. Contamination with another substrate DNA. 

The sample DNA may contain a mixture of two or more different DNAs. Prepare new sample of DNA. 

E.coli grow cells and purify plasmid 

 

 

 

2.4. Incomplete DNA digestion see 

2.5. Gel shift see 

2.6. Unexpected recognition sites in template DNA. 

Newly generated target sites in constructed DNA may be overlooked. Re-check your DNA sequence and 

cloning strategy. Refer to Tables Table 1.21, Table 1.22, Table 1.23 or Table 1.24 for Newly Generated



3. Diffused DNA bands 

3.1. Gel shift. 

Enzyme that remains bound to the substrate DNA will affect the electrophoretic mobility of the digestion products. 

FastDigest 

conventional restriction enzymes AarI, AloI, BseXI, BveI, CseI, Eco57I, EcoRII, FaqI, GsuI, Lsp1109I, LweI, MboII, 

MnlI, SchI, TauI and TscAI are particularly prone to remaining bound to the substrate DNA. This will result in a 

see 

presence of 

M 1 2 3 4 

M – GeneRuler 

1 – 0.5 μg 

2 – 0.5 μg 

3 – 0.5 μg DNA digested with FastDigest 

 

4 – 0.5 μg DNA digested with FastDigest FokI, probe prepared for loading with 

 

3.2. Contaminated reagents. 

Any restriction digestion reaction components may become contaminated with nucleases due to improper 

handling or storage. Nuclease contamination causes DNA degradation, which appears as diffused DNA bands on 

a gel. 

Perform four control reactions in order to check for the nuclease contamination: 

I – without restriction enzyme, II – with a new vial of buffer, III – without restriction enzyme, with a new vial of 

buffer, IV 

 

 

improper handling. Use a new vial of enzyme. 

 

DNA degradation. Use a new vial of buffer. Store all buffers at -20°C. 

 

given above. 

 

handled water will cause DNA degradation. Use commercially available nuclease-free molecular biology grade 

 


234 


Thermo Scientific DNA/RNA 

Modifying Enzymes 

Quality Control .................................................................................................................236 

Quality Control Assays ...................................................................................................236 

Storage and Shipping ..................................................................................................... 237 

Description of Icons ....................................................................................................... 237 

Ligases ..............................................................................................................................238 

T4 DNA Ligase ..............................................................................................................239 

T4 RNA Ligase............................................................................................................... 240 

Phosphatase and Kinase ............................................................................................... 241 

FastAP Thermosensitive Alkaline Phosphatase ...........................................................242 

T4 Polynucleotide Kinase (T4 PNK) .............................................................................. 243 

DNA Polymerases ............................................................................................................ 244 

phi29 DNA Polymerase ................................................................................................ 245 

DNA Polymerase I ........................................................................................................ 246 

Klenow Fragment ......................................................................................................... 247 

Klenow Fragment, exo – ................................................................................................ 248 

T4 DNA Polymerase ...................................................................................................... 249 

T7 DNA Polymerase .....................................................................................................250 

Terminal Deoxynucleotidyl Transferase (TdT) ............................................................ 251 

Terminal Transferase ................................................................................................... 252 

Bsm DNA Polymerase, Large Fragment .....................................................................253 

Deoxyribonucleases (DNases) .....................................................................................254 

DNase I, RNase-free .....................................................................................................255 

Endonuclease IV, E.coli (Endo IV) .................................................................................256 

Endonuclease V, T.maritima (Endo V) .......................................................................... 257 

Exonuclease I (Exo I) .....................................................................................................258 

Exonuclease III (Exo III) ................................................................................................. 259 

Lambda Exonuclease ...................................................................................................260 

Ribonucleases (RNases) ................................................................................................ 261 

RNase A, DNase and Protease-free ............................................................................262 

RNase T1 .......................................................................................................................263 

RNase A/T1 Mix ............................................................................................................264 

RNase I ..........................................................................................................................265 

RNase H .........................................................................................................................266 

PowerCut Dicer ............................................................................................................ 267 

Sugar Nonspecific Nucleases.......................................................................................268 

Micrococcal Nuclease .................................................................................................268 

S1 Nuclease ..................................................................................................................269 

Other Products ................................................................................................................. 270 

Proteinase K (recombinant), PCR grade ......................................................................... 270 

RiboLock RNase Inhibitor ............................................................................................ 271 

Pyrophosphatase, Inorganic (from yeast) .................................................................... 272 

Uracil-DNA Glycosylase (UDG, UNG) ............................................................................ 273 


2.1. Activity of DNA/RNA modifying enzymes in Thermo Scientific buffers ......................... 274 

2.2. Dilution of DNA/RNA modifying enzymes .................................................................. 275 

Learn about 


modifying enzymes for 

cloning applications at: 


Purify 

Digest 

Amplify 

Modify 


236 


All Thermo Scientific DNA/RNA modifying 

enzymes are produced in class D clean 

room facilities under ISO 9001:2008 quality 

management system and are subjected to 

extensive quality control. 

Quality Control Assays 

Activity Assays 

Activity unit definitions for Thermo Scientific 

DNA/RNA modifying enzymes are those 

commonly used in molecular biology. Activity 

unit definitions and activity assay conditions are 

provided in the catalog entry for each product. 

Reaction conditions may differ for specific 

research applications. 


The LO test is a unique quality test designed 

and applied for detection of endo- and 

exodeoxyribonucleases. This test is able 

to detect trace amounts of contaminating 

activities and is the most sensitive assay in the 

industry. The assay is performed in reaction 

buffer containing a particular enzyme or 

other protein of interest and 5’-[ 32 P]-labeled 

synthetic oligonucleotides (single-stranded 

and double-stranded). After incubation under 

the appropriate conditions, reaction products 

are separated on a polyacrylamide gel and 

then analyzed by phosphorimaging. The 

product passes the LO quality control test 

if there is no degradation of both the singlestranded 

oligonucleotide and double-stranded 

oligonucleotide. 

Double-stranded 

Endodeoxyribonuclease Assay 

The assay is performed in a 50 μl reaction 

mixture containing reaction buffer, enzyme and 

1 μg of supercoiled pUC19 DNA or X174 RF1 

DNA. After incubation under the appropriate 

conditions, the DNA is analyzed on a 1% 

agarose gel. The product passes this quality 

control test if neither nicked DNA nor linear DNA 

is detected. 


Our products are monitored for unit 

concentration accuracy, the absence of 

contaminant activities (nucleases, proteases) 

and their performance in specific functional 

tests. 

Single-stranded 

Endodeoxyribonuclease Assay 


mixture containing reaction buffer, enzyme 

and 1 μg of covalently closed circular singlestranded 

DNA of M13mp19. After incubation 

under appropriate conditions, DNA is analyzed 

on a 1% agarose gel. The product passes this 

quality control test if no decrease in the amount 

of closed circular DNA is observed. 

Exodeoxyribonuclease Assay 



1 μg of either the lambda DNA or plasmid DNA 

fragments. After incubation under appropriate 

conditions, the DNA is analyzed on an agarose 

gel. The product passes this quality control test 

if the DNA fragments are not degraded. 

Ribonuclease Assay I 


mixture containing reaction buffer, enzyme 

and 1 μg of [ 3 H]-labeled RNA. After incubation 

under the appropriate conditions, the RNA is 

precipitated with trichloroacetic acid and the 

radioactivity of the supernatant is determined. 

RNase contamination is specified as a 

percentage of the total RNA released into the 

acid soluble fraction. 

A warranty is assigned and an expiry date 

is listed both on the product label and in the 

Certificate of Analysis supplied with each 

product. Product lots are monitored to ensure 

quality specifications are met up to the expiry 

date. 

Ribonuclease Assay II 



160 ng of 2 kb synthetic RNA. After incubation 

under the appropriate conditions, the RNA 

integrity is analyzed on a 1% agarose gel. The 

product passes this quality control test if no 

degradation of RNA is observed. 

Protease Assay 


mixture containing 10 mM Tris-HCl buffer 

(pH 7.5), enzyme and 200 μg of azocasein. 

After incubation under the appropriate 

conditions, the reaction is terminated with 

trichloroacetic acid and the absorbance of 

the supernatant is measured at 400 nm. 

The product passes this quality control test if 

azocasein is not degraded. 

E.coli Genomic DNA Assay 

Contamination of enzymes with E.coli genomic 

DNA is tested by qPCR with primers and probes 

specific to 23S rRNA gene. 

Functional Assays 

Specific assays performed for a particular 

modifying enzyme or other protein are indicated 

both in the product entry and in the Certificate 

of Analysis supplied with each product. 

Note 

The conditions for Quality Control Assays for the enzymes are 

described in the Certificate of Analysis.


DNA/RNA modifying enzymes should be stored 

at -20°C. 

Enzymes may freeze during shipment on dry 

ice. This does not affect their quality, as all 

Thermo Scientific enzymes are tested 100% 

Description of Icons 

20’ 

active after at least three freeze-thaw cycles. 


shipped on blue ice, because their quality is not 

affected by short exposure to 4°C. 

FastDigest Buffer. Denotes 100% activity of the enzyme in the FastDigest or FastDigest 

Green Buffer. 

Five Buffer System. Letters in the buffer icons correspond to the codes of the Five Buffer System: 

B (blue), G (green), O (orange), R (red) and Tango respectively. Denotes 100% activity of the enzyme 

in the indicated buffer. 

Thermal Inactivation. Denotes the possibility and conditions for thermal inactivation. 

Thermal Inactivation in the presence of EDTA. Denotes the possibility and conditions of thermal 

inactivation in the presence of EDTA. 

EDTA Inactivation. Denotes the possibility of inactivation by EDTA. 

High Concentration enzyme format is available. 

Low Concentration enzyme format is available. 

Recombinant Protein. Purified from an E.coli strain carrying a cloned gene encoding the protein. 

LO Certified. Product has been tested in the Labeled Oligonucleotide (LO) test, see p.236. 

NEW for 2012. 


238 

Ligases 

Table 2.1. Properties of ligases. 

Ligase Applications Substrate Reaction catalyzed Inactivation Cat. # Page 

T4 DNA Ligase 

T4 RNA Ligase 

ss – single-stranded, 

ds – double-stranded. 


 

termini. 

 

adaptors to dsDNA. 

 

DNA-RNA hybrids. 

 

 

 

 

3’,5’-bis [- 32 P] phosphate. 

 

 

oligodeoxyribonucleotides. 

 

 

single-stranded cDNAs for 5’ RACE. 

 

primers for PCR. 

dsDNA 

dsRNA 

dsDNA-RNA 

hybrids 

ds poly- and 

oligonucleotides 

ssDNA 

ssRNA 

poly- and 


ATP dependent intra- and 

intermolecular formation of a 

phosphodiester bond between 

juxtaposed 5’-phosphate and 

3’-hydroxyl termini in duplex 

DNA or RNA. 

ATP dependent intra- and 

intermolecular formation 

of phosphodiester bonds 

between 5’-phosphate and 

3’-hydroxyl termini of poly- 

and oligonucleotides, ssRNA 

and ssDNA. 

65°C, 10 min or 

70°C, 5 min 

EL0011/2/3/4/6 239 

70°C, 10 min EL0021 240

T4 DNA Ligase 

#EL0014 200 u (5 u/μl) 


10X T4 DNA Ligase Buffer 0.5 ml 

50% PEG 0.3 ml 

#EL0011 1000 u (5 u/μl) 

#EL0016 LC, 2x500 u (1 u/μl) 


10X T4 DNA Ligase Buffer 1.5 ml 

50% PEG 1.5 ml 

#EL0012 5x1000 u (5 u/μl) 

#EL0013 HC, 5000 u (30 u/μl) 


10X T4 DNA Ligase Buffer 5x1.5 ml 

50% PEG 5x1.5 ml 

10X T4 DNA Ligase Buffer 

#B69 1.5 ml 

Related Products 

Rapid DNA Ligation Kit p.288 

PCR Cloning Kit p.279 

InsTAclone PCR Cloning Kit p.281 

TransformAid Bacterial Transformation Kit p.289 

Nicking Enzymes p.155 

T4 DNA Polymerase p.249 

Klenow Fragment p.247 

T7 DNA Polymerase 

FastAP Thermosensitive Alkaline 

p.250 

Phosphatase p.242 

T4 Polynucleotide Kinase (T4 PNK) p.243 

6X DNA Loading Dye & SDS Solution p.373 

ATP p.411 

Water, nuclease-free p.420 

References 

1. Rossi, R., et al., Functional characterization of the T4 

DNA Ligase: a new insight into the mechanism of action, 

Nucleic Acids Res., 25, 2106-2113, 1997. 

2. Cherepanov, A.V., et al., Binding of nucleotides by T4 

DNA Ligase and T4 RNA Ligase: optical absorbance and 

 

3. Nilsson, M., et al., RNA-templated DNA ligation for 

transcript analysis, Nucleic Acids Res., 29, 

578-581, 2001. 

4. Weiss, B., et al., Enzymatic breakage and joining of 

 

4543-4555, 1968. 

5. Pheiffer, B.H., Zimmerman, S.B., Polymer-stimulated 

ligation: enchanced blunt- or cohesive-end ligation of DNA 

or deoxyribo-oligonucleotides by T4 DNA ligase in polymer 

solutions, Nucleic Acids Res., 11, 7853-7871, 1983. 

Description 

T4 DNA Ligase catalyzes the formation of a 

phosphodiester bond between juxtaposed 

5’-phosphate and 3’-hydroxyl termini in duplex 

DNA or RNA. The enzyme repairs single-strand 

nicks in duplex DNA, RNA or DNA/RNA hybrids, 

joins DNA fragments with either cohesive or 

blunt termini (1, 2). 

The T4 DNA Ligase requires ATP as cofactor. 

Features 

Fast – sticky-end ligation is completed in 

10 min at room temperature. 

Active in buffers for restriction enzymes, PCR 

and RT (when supplemented with ATP). 

Supplied with PEG solution for efficient bluntend 

ligation. 

Applications 

Cloning of restriction enzyme generated DNA 

fragments. 

Cloning of PCR products. 

 

linkers or adaptors to DNA. 

Site-directed mutagenesis. 

Amplified fragment length polymorphism 

(AFLP). 

Ligase-mediated RNA detection (3). 

Nick repair in duplex DNA, RNA or DNA/RNA 

hybrids. 

Self-circularization of linear DNA. 

Concentration 

1 Weiss u/μl = 200 CEU*/μl 



* One CEU is defined as the amount of enzyme required to give 

50% ligation of HindIII fragments of lambda DNA in 30 min 

at 16°C. 

Source 

E.coli cells with a cloned gene 30 of 

bacteriophage T4. 

Molecular Weight 

55.3 kDa monomer. 

Definition of Activity Unit 

One Weiss unit of the enzyme catalyzes the 

conversion of 1 nmol of [ 32PPi] into Noritadsorbable 

form in 20 min at 37°C (4). One 

Weiss unit is equivalent to approximately 200 

cohesive end ligation units (CEU)*. 

Enzyme activity is assayed in the following mixture: 

66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl2, 0.066 mM ATP, 10 mM DTT, 3.3 μM [ 32PPi]. Storage Buffer 

The enzyme is supplied in: 

20 mM Tris-HCl (pH 7.5), 1 mM DTT, 

50 mM KCl, 0.1 mM EDTA 0.015% ELUGENT 

Detergent and 50% (v/v) glycerol. 

10X T4 DNA Ligase Buffer (#B69) 

400 mM Tris-HCl, 100 mM MgCl2, 100 mM 

DTT, 5 mM ATP (pH 7.8 at 25°C). 

50% PEG Solution 

50% (w/v) polyethylene glycol 4000. 

Inhibition and Inactivation 

 

by NaCl or KCl at >200 mM. 

Inactivated by heating at 65°C for 10 min or 

70°C for 5 min. 

Note 

 

the rate of ligation of blunt-end DNA (5). The 

suggested concentration of PEG 4000 in the 

reaction mixture is 5% (w/v). 

 

in a band shift in agarose gels. To avoid this, 

incubate samples with 6X DNA Loading Dye 

& SDS Solution (#R1151) at 70°C for 5 min 

or 65°C for 10 min and chill on ice prior to 


 

should not exceed 10% of the competent 

cell volume in the transformation process. 

 

T4 DNA Ligase from the ligation mixture by 

spin column or chloroform extraction. The 

extracted DNA can be further precipitated 

with ethanol. 


» 2.1. Activity of DNA/RNA modifying 

enzymes in Thermo Scientific buffers p.274 

» 3.1.1. Sticky-end ligation p.294 

3.1.2. Blunt-end ligation p.294 

» 3.1.3. Self-circularization of linear DNA p.294 

» 3.1.4. Linker ligation p.294 

» 3.1.5. Analysis of ligation products by 

agarose gel electrophoresis p.294 


240 

T4 RNA Ligase 

#EL0021 1000 u (10 u/μl) 


10X Reaction Buffer 0.2 ml 

1mg/ml BSA Solution 0.2 ml 


RiboLock RNase Inhibitor p.271 

TranscriptAid T7 High Yield Transcription Kit p.328 

RNA Polymerases pp.330-331 

RNase H 


p.266 



RiboRuler RNA Ladders p.386 

ATP p.411 

Bovine Serum Albumin (BSA) p.422 



» 6.1.6. RNA 3’-end labeling by ligation p.350 


Description 

T4 RNA Ligase catalyzes the ATP-dependent 

intra- and intermolecular formation of 

phosphodiester bonds between 5’-phosphate 

and 3’-hydroxyl termini of oligonucleotides, 

single-stranded RNA and DNA. 

The minimal substrate is a nucleoside 

3’,5’-biphosphate in intermolecular reaction 

and oligonucleotide of 8 bases in 

intramolecular reaction. 

Applications 

RNA 3’-end labeling with cytidine 3’,5’-bis 

[-32P] phosphate (1). 

 

Synthesis of oligoribonucleotides and 

oligodeoxyribonucleotides (3, 4). 

Specific modifications of tRNAs (5). 

Oligodeoxyribonucleotide ligation to 

single-stranded cDNAs for 5’ RACE (Rapid 

Amplification of cDNA Ends) (6). 

Site-specific generation of composite primers 

for PCR (7). 

Concentration 

10 u/μl 

Source 






One unit of the enzyme catalyzes the conversion 

of 1 nmol of 5’-[ 32P]-(A) 12-18 to a phosphataseresistant 

form in 30 min at 37°C. 

Enzyme activity is assayed in the following 

mixture: 50 mM Tris-HCl (pH 7.5), 10 mM 

MgCl2, 10 mM DTT, 1 mM ATP, 10 μM 5’-[ 32P]- (A) 12-18 (10 μM in 5’-termini). 



20 mM Tris-HCl (pH 7.5), 1 mM DTT, 

50 mM KCl, 

0.1 mM EDTA, 0.015% (v/v) ELUGENT 

Detergent and 50% (v/v) glycerol. 

10X Reaction Buffer 

500 mM Tris-HCl (pH 7.5 at 25°C), 

100 mM MgCl2, 100 mM DTT, 10 mM ATP. 


 

modifying reagents (8). 

 

Note 

 

reaction mixture is 0.1 mg/ml. 

References 

1. Uhlenbeck, O.C., Gumport, R.I., T4 RNA ligase, The 

Enzymes (Boyer, P.D., Ed.), Academic Press Inc., New York, 

15B, 31-60, 1982. 

2. Middleton, T., et al., Synthesis and purification of 

oligoribonucleotides using T4 RNA ligase and reverse-phase 

chromatography, Anal. Biochem., 144, 110-117, 1985. 

3. Brennan, C.A., et al., Using T4 RNA ligase with DNA 

substrates, Meth. Enzymol., 100, 38-52, 1983. 

4. Tessier, D.C., et al., Ligation of single-stranded 

oligodeoxyribonucleotides by T4 RNA ligase, Anal. 

Biochem., 158, 171-178, 1986. 

5. Heckler, T.G., et al., T4 RNA ligase mediated preparation of 

novel “chemically misacylated’’ tRNA Phe s, Biochemistry, 23, 

1468-1473, 1984. 

6. 

single-stranded cDNAs: a new tool for cloning 5’-ends 

of mRNAs and for constructing cDNA libraries by in vitro 

amplification, Nucleic Acids Res., 19, 5227-5232, 1991. 

7. Kaluz, S., et al., Enzymatically produced composite 

primers: an application of T4 RNA ligase-coupled primers 

to PCR, BioTechniques, 19, 182-186, 1995. 

8. Eun, H. M., Enzymology Primer for Recombinant DNA 

Technology, Academic Press. Inc., 1996.

Phosphatase and Kinase 

Table 2.2. Properties of phosphatase and kinase. 

Phosphatase / 

Kinase 

FastAP 

Thermosensitive 

Alkaline 

Phosphatase 

T4 Polynucleotide 

Kinase (T4 PNK) 

Applications Substrates 

Dephosphorylation 

of DNA, RNA and 

oligonucleotides. 

Dephosphorylation of 

vector DNA to avoid 

recircularization. 

PCR product clean-up 

before sequencing. 

Dephosphorylation of 

proteins. 

Phosphorylation of PCR 

products prior to ligation. 

Phosphorylation of PCR 

primers. 

5’-phosphorylation of 

oligonucleotide and DNA 

or RNA prior to ligation. 

Labeling 5’-termini of 

nucleic acids. 

 

primers. 

Labeling of DNA and 

RNA markers. 

Detection of DNA 

modification by the 

[ 32 P]-postlabeling assay. 

Removal of 3’-phosphate 

group. 

5’- and 

3’-phosphorylated 

DNA/RNA, 


dNTPs/NTPs, 

phosphorylated 

proteins 

ATP*, DNA/RNA, 

oligonucleotides, 

3’-NMP, 


DNA/RNA, 

3’-P-oligonucleotides, 

deoxynucleoside 

3’-monophosphates, 

deoxynucleoside 

3’-diphosphates 

Reaction 

catalyzed 

Release of 

phosphate group 

Transfer of the 

-phosphate from 

ATP* to the 5’-OH 

group of DNA/RNA, 


3’-NMP, 

exchange of 

phosphate group 

between 

5’-P-oligo-/ 

polynucleotides 

and ATP (in the 

presence of ADP), 

release of 

3’-phosphate group 

ss – single-stranded, 

ds – double-stranded, 

* although ATP is routinely used as a phosphate donor, CTP, UTP, GTP, dATP, dTTP are equally effective. 

Reaction 

products 

5’, 3’-OH ends of 

DNA/RNA, 


deoxynucleosides/ 

nucleosides, 

dephosphorylated 

proteins 

5’-phosphory- 

lated DNA/RNA, 

5’-P-oligonucleotides, 

3’, 5’-NDP, 

3’-dephosphorylated 

DNA/RNA 


deoxynucleosides 

Reaction 

conditions 

Inactivation Cat. # Page 

37°C, 10 min 75°C, 5 min EF0651/2 242 

37°C, 20 min 

75°C, 10 min 

or addition of 

EDTA 

EK0031/2 243 


242 

FastAP Thermosensitive Alkaline Phosphatase 

#EF0651 1000 u (1 u/μl) 

(for 1000 rxns) 


10X FastAP Buffer 2x1.5 ml 

#EF0652 5x1000 u (1 u/μl) 




#EF0654 300 u (1 u/μl) 






Exonuclease I (Exo I) p.258 

T4 DNA Ligase p.239 

T4 RNA Ligase p.240 


6X DNA Loading Dye & SDS Solution p.373 



» 2.1. Activity of DNA/RNA modifying enzymes 

in Thermo Scientific buffers p.274 

» 3.4. Dephosphorylation of DNA and RNA p.295 

» 3.4.2. Fast simultaneous plasmid vector 

linearization and dephosphorylation p.295 

» 4.8. PCR product clean-up prior to 

sequencing p.323 

» 6.1.3. Radiolabeling of RNA Ladders 

by T4 PNK p.349 


Description 


catalyzes the release of 5’- and 3’-phosphate 

groups from DNA, RNA and nucleotides. 

This enzyme also removes phosphate groups 

from proteins. 

FastAP is a novel alkaline phosphatase, which is 

active in all Thermo Scientific restriction enzyme 

buffers as well as in PCR and RT buffers. It 

dephosphorylates all types of DNA ends in 

10 min at 37°C. The enzyme is inactivated in 

5 min at 75°C eliminating the need to remove 

the enzyme from the reaction mixture 

prior to ligation. 

Features 

Recombinant enzyme. 

Fast dephosphorylation – 10 min at 37°C. 

Fast thermoinactivation – 5 min at 75°C. 

Active in Thermo Scientific buffers for 

restriction enzymes PCR and RT buffers. 

Does not require additives or buffer change. 

Single protocol for all types of DNA ends. 

Active on nucleotides. 

Applications 

Dephosphorylation of cloning vector DNA to 

prevent recircularization during ligation. 

Simultaneous digestion and 

dephosphorylation of vector DNA. 

PCR product clean-up: nucleotide 

degradation prior to sequencing of PCR 

product. 

Dephosphorylation of nucleic acid 5’-termini 

prior to labeling with T4 Polynucleotide Kinase. 

Other applications where dephosphorylation 

of DNA and RNA substrates is necessary. 

Protein dephosphorylation. 

Concentration 

1 u/μl 

Source 

E.coli cells with a cloned bacterial AP gene. 


One unit is amount of the enzyme required to 

dephosphorylate 1 μg of linearized pUC57 DNA 

5’-termini in 10 min at 37°C in FastAP buffer. 



20 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 0.1 mM ZnCl2, 0.1% (v/v) Triton X-100, 

50% (v/v) glycerol. 

10X FastAP Buffer 


50 mM MgCl2, 1 M KCl, 0.2% (v/v) Triton X-100, 

1 mg/ml BSA. 


 

 

Note 

Thermosensitive Alkaline 

Phosphatase to DNA may result in a band 

shift in agarose gels. To avoid this, incubate 

samples with 6X DNA Loading Dye & SDS 

Solution (#R1151) at 65°C for 10 min and 

chil on ice prior to electrophoresis. 

Thermosensitive Alkaline 

Phosphatase is active in all restriction 

enzyme buffers and may be added directly 

to digested DNA. Heat inactivation of the 

restriction enzyme before dephosphorylation 

reaction is not necessary. 

2 

1 

0 

0 5 10 15 

Incubation time, min 

% of activity remaining 100 

Thermoinactivation curve for Thermo Scientific 


at 75°C. 

The DNA dephosphorylation reaction mixture (200 u/ml 

phosphatase) was incubated for 30 min at 37°C. 

Aliquots were heated at 75°C for the time indicated 

on the graph. The remaining activity was measured by 

p-NPP assay.

T4 Polynucleotide Kinase (T4 PNK) 

#EK0031 500 u (10 u/μl) 


10X Reaction Buffer A 0.4 ml 

10X Reaction Buffer B 0.2 ml 

24% PEG Solution 0.2 ml 

#EK0032 2500 u (10 u/μl) 


10X Reaction Buffer A 2x1 ml 

10X Reaction Buffer B 1 ml 

24% PEG Solution 1 ml 




GeneRuler DNA Ladders p.358 


ATP p.411 

0.5 M EDTA, pH 8.0 p.421 


References 

1. Berkner, K.L., Folk, W.R., Polynucleotide kinase exchange 

 

2. Richardson, C.C., Bacteriophage T4 polynucleotide kinase, 

The Enzymes (Boyer, P.D., Ed.), Academic Press, San 

Diego, 14, 299-314, 1981. 

3. 

A Laboratory Manual, the Third edition, Cold Spring Harbor 

Laboratory Press, Cold Spring Harbor, New York, 2001. 

4. Current Protocols in Molecular Biology, vol. 1 (Ausubel, 

 

York, 3.10.2-3.10.5, 1994-2004. 

5. Phillips, D.H., Detection of DNA modifications by the 

32 P-postlabelling assay, Mutation Res., 378, 1-12, 1997. 

6. Keith, G., Dirheimer, G., Postlabeling: a sensitive method 

for studying DNA adducts and their role in carcinogenesis, 

Curr. Opin. Biotechnol., 6, 3-11, 1995. 

7. Harrison, B., Zimmerman, S.B., T4 polynucleotide kinase: 

macromolecular crowding increases the efficiency of 

reaction at DNA termini, Anal. Biochem., 158, 

307-315, 1986. 

Description 

T4 Polynucleotide Kinase (T4 PNK) catalyzes 

the transfer of the -phosphate from ATP to the 

5’-OH group of single- and double-stranded 

DNAs and RNAs, oligonucleotides or nucleoside 

3’-monophosphates (forward reaction). The 

reaction is reversible. In the presence of ADP T4 

Polynucleotide Kinase exhibits 5’-phosphatase 

activity and catalyzes the exchange of 

phosphate groups between 5’-P-oligo-/polynucleotides 

and ATP (exchange reaction) (1). 

The enzyme also displays 3’-phosphatase 

activity (2). 

Features 


restriction enzymes, reverse transcriptases 

and T4 DNA Ligase. 

Applications 

Labeling 5’-termini of nucleic acids (3, 4) to 

be used as: 

– probes for hybridization, 

– probes for transcript mapping, 

– markers for gel-electrophoresis, 

– primers for DNA sequencing, 

– primers for PCR. 

5’-phosphorylation of oligonucleotides, PCR 

products, other DNA or RNA prior to ligation. 

Phosphorylation of PCR primers. 

Detection of DNA modification by the 

[ 32P]-postlabeling assay (5, 6). 

Removal of 3’-phosphate groups (2). 

Concentration 

10 u/μl 

Source 

E.coli cells with a cloned pseT gene of 



The enzyme is a homotetramer. It consists of 

four identical subunits of 28.9 kDa. 

x10 6 dpm/pmol 5’-ends 

7 

6 

5 

4 

3 

2 

1 

0 

5’-overhang 

ends of DNA 

forward reaction in buffer A 

exchange reaction in buffer B 

blunt ends 

of DNA 

5’-recessed 

ends of DNA 

Labeling efficiency of DNA containing different 

types of 5’-ends. 


One unit of the enzyme transfers 1 nmol of 

-phosphate from ATP to 5’-OH DNA in 

30 min at 37°C. 


mixture: 100 mM Tris-HCl (pH 8.0), 

10 mM MgCl 2, 5 mM DTT, 0.5 mM 5’-OH DNA, 

0.05 mM ATP and 0.1 MBq/ml [- 33 P]-ATP. 



20 mM Tris-HCl (pH 7.5), 25 mM KCl, 

0.1 mM EDTA, 2 mM DTT and 


10X Reaction Buffer A (for forward reaction) 


100 mM MgCl2, 50 mM DTT, 1 mM spermidine. 

10X Reaction Buffer B (for exchange reaction) 

500 mM imidazole-HCl (pH 6.4 at 25°C), 

180 mM MgCl2, 50 mM DTT, 1 mM spermidine 

and 1 mM ADP. 

24% PEG Solution 

24% (w/v) polyethylene glycol 6000. 


 

and ammonium ions, KCl and NaCl at a 

concentration higher than 50 mM. 

Inactivated by heating at 75°C for 10 min 

or by addition of EDTA. 

Note 

 

improve the rate and efficiency of the 

phosphorylation reaction (7). PEG is used 

in the exchange reaction. 

 

ammonium ions, use sodium 

acetate to precipitate DNA prior to 

phosphorylation (1, 2). 




» 3.3. Phosphorylation of DNA p.295 

» 6.1.1. DNA 5’-end labeling by T4 PNK 

in the exchange reaction p.349 

» 6.1.2. DNA/RNA 5’-end labeling by T4 PNK 

in the forward reaction p.349 

» 6.1.3. Radiolabeling of RNA Ladders 



244 

DNA Polymerases 

Table 2.3. Properties of mesophilic DNA polymerases. 

Polymerase Applications Template 

phi29 DNA Polymerase 

DNA Polymerase I 

Klenow Fragment 

Klenow Fragment, exo – 



Terminal 

Deoxynucleotidyl 

Transferase (TdT) 


 

 

 

 

 

 

In situ genotyping. 

 

 

 

 

 

 

 

 

 

synthetic oligonucleotides. 

 

 

 

 

 

 

 

overhangs. 

 

 

 

products. 

Purification of covalently closed circular DNA 

by removal of residual genomic DNA. 

 

 

 

mutagenesis. 

 

 

In situ detection of DNA fragmentation 

associated with apoptosis. 

 

heteropolymers. 

 

with any type of 3’-OH terminus. 

 

 

In situ localization of apoptosis. 

 

(LAMP). 

 

 

 

 

3’5’ 

exonuclease 

5’3’ Strand 


exonuclease displacement 

DNA ++++ – ++++ 65°C, 10min EP0091/2/4 245 

DNA ++ + – 75°C, 10min EP0041/2 246 

DNA ++ – ++ 75°C, 10min EP0051/2/4 247 

DNA – – ++ 75°C, 10min EP0421/2 248 

DNA ++++ – – 75°C, 10min EP0061/2 249 

DNA ++++ – – 75°C, 10min EP0081 250 

ssDNA 

dsDNA 

EN0361/2 251 

ssRNA 

– – NA 70°C, 10min 

Terminal Transferase* dsRNA 

F-203S/L 252 

Bsm DNA Polymerase 

– no activity, + active. 

NA – not applicable. 

* – formerly manufactured by Finnzymes OY, now part of Thermo Fisher Scientific. 

DNA – – ++++ 80°C, 10 min EP0691 253 

Find Thermo Scientific 

DNA polymerases and reverse 

transcriptases for PCR and 

qPCR applications at: 

www.thermoscientific.com/pcr

phi29 DNA Polymerase 

#EP0091 2.5 μg (0.1 μg/μl) 

250 u (10 u/μl) 



#EP0092 10 μg (0.1 μg/μl) 

1000 u (10 u/μl) 


10X Reaction Buffer 1 ml 

#EP0094 50 μg (0.1 μg/μl) 

5000 u (10 u/μl) 


10X Reaction Buffer 5x1 ml 


GeneRuler High Range DNA Ladder p.358 

Conventional Lambda DNA Markers p.368 

Pyrophosphatase, Inorganic (from yeast) p.272 

dNTP Set p.408 

dNTP Mix, 10 mM each p.408 


References 

1. Blanco, L., et al., Highly Efficient DNA Synthesis by the phage phi29 

 

Chem., 264, 8935-8940, 1989. 

2. Garmendia, C., et al., The bacteriophage phi29 DNA polymerase, a 

 

3. Lagunavicius, A., et al., Duality of polynucleotide substrates for 

Phi29 DNA polymerase: 3’5’ RNase activity of the enzyme, RNA, 

14, 503-513, 2008. 

4. Skerra, A., Phosphorothioate primers improve the amplification of 

DNA sequences by DNA polymerases with proofreading activity, 


5. 

Chem., 268, 2719-2726, 1993. 

6. Lizardi, P. M., et al., Mutation detection and single-molecule 

counting using isothermal rolling-circle amplification, Nat. Genet., 

19, 225-232, 1998. 

7. Simmel, F.C., et al., Periodic DNA nanotemplates synthesized by 

rolling circle amplification, Nano Letters, 4, 719-722, 2005. 

8. Dean, F.B., et al., Comprehensive human genome amplification 

using multiple displacement amplification, Proc. Natl. Acad. Sci. 

USA, 99, 5261-5266, 2002. 

9. Wojnowski, L., et al., Genome-wide single-nucleotide polymorphism 

arrays demonstrate high fidelity of multiple displacement-based 

whole-genome amplification, Electrophoresis, 26, 710-715, 2005. 

10. 

amplification in a 5 cM STR genome-wide scan, Nucleic Acids Res., 

13, e1191, 2005. 

11. Gadkar V, Rillig MC., Suitability of genomic DNA synthesized 

by strand displacement amplification (SDA) for AFLP analysis, 

genotyping single spores of arbuscular mycorrhizal (AM) fungi, 

 

12. Sgaramella, V., et al., Towards the analysis of the genomes of 

single cells, Further characterisation of the multiple displacement 

amplification, Gene 372, 1-7, 2006. 

13. Matsunaga, T., et al., Whole-metagenome amplification of a 

microbial community associated with scleractinian coral by multiple 

displacement amplification using phi29 polymerase, Environmental 

Microbiology, 8, 1155-1163, 2006. 

14. Andersen, P.S., et al., Whole genome amplification on DNA from 

filter paper blood spot samples, an evaluation of selected systems, 

Genetic Testing, 11, 2007 

15. Blanco, L., et al., Terminal protein – primed DNA amplification, Proc. 

Natl. Acad. Sci. USA, 91, 12198-12202, 1994. 

16. Lagunavicius, A., et al., Novel application of phi29 DNA polymerase: 

RNA detection and analysis in vitro and in situ by target RNA-primed 

RCA, RNA, 12, 765-771, 2009. 

17. Larsson, C., et al., In situ genotyping individual DNA molecules by 

target-primed rolling-circle amplification of padlock probes, Nat. 

Methods, 1, 227-232, 2004. 

Description 

phi29 DNA Polymerase is a highly processive 

polymerase (more than 70 kb) featuring strong 

strand displacement activity which allows for 

highly efficient isothermal DNA amplification 

(1). phi29 DNA Polymerase also demonstrates 

3’5’ exonuclease (proofreading) activity 

acting preferentially on single-stranded DNA (2) 

or RNA (3). Therefore 3’-modified exonuclease 

resistant primers are highly recommended 

for use in reactions containing phi29 DNA 

polymerase (4). 

Features 

Highest processivity and strand displacement 

activity among known DNA polymerases – 

more than 70 kb long DNA stretches can be 

synthesized (1). 

Highly accurate DNA synthesis (5). 

Extremely high yields of amplified DNA even 

from minimal amounts of template. 

Amplification products can be directly used 

in downstream applications (PCR, restriction 

digestion, SNP genotyping, etc.). 

Applications 

Rolling circle amplification (RCA) (6): 

generation of periodic DNA nanotemplates (7). 

Multiple displacement amplification (MDA) (8). 

Unbiased amplification of whole genome 

(WGA): 

– amplification of DNA for SNP (9) and STR 

(10) detection, 

– cell-free amplification of DNA from single 

cells (11, 12), pathogenic organisms or 

metagenomes (13), 

– amplification of DNA from filter paper 

blood spot samples (14). 

DNA template preparation for sequencing. 

Protein-primed DNA amplification (15). 

RNA-primed DNA amplification (16). 

In situ genotyping with padlock probes (17). 

Recombination based-cloning (18). 

Cell-free cloning of lethal DNA (19). 

Concentration 

0.1 μg/μl; 10 u/μl 

Source 

E.coli cells with a cloned gene 2 of Bacillus 

subtilis phage phi29. 

18. 

products are compatible with recombination-based cloning, 

BioTechniques, 42, 706-708, 2007. 

19. Hutchison CA et al., Cell-free cloning using phi29 DNA polymerase, 

Proc. Natl. Acad. Sci. U S A 29,102, 17332-17336, 2005. 

20. Blanco, L., Salas, M., Effect of aphidicolin and nucleotide analogs of 

on the phage phi29 DNA polymerase, Virology, 153, 

179-187, 1986. 



Specific Activity 

100,000 u/mg 


One unit of the enzyme catalyzes the 

incorporation of 0.5 pmol of dCMP into a 

polynucleotide fraction (adsorbed on DE-81) in 

10 min at 30°C. 



10 mM MgCl2, 1 mM DTT, 0.01 mg/ml lambda DNA/HindIII, 

0.2 μM dCTP including [ 3H]-dCTP, 0.2 mM dATP, 

0.2 mM dGTP and 0.2 mM dTTP. 


The enzyme is supplied in: 50 mM Tris-HCl 

(pH 7.5), 0.1 mM EDTA, 1 mM DTT, 100 mM 

KCl, 0.5% (v/v) Nonidet P40, 0.5% (v/v) Tween 

20 and 50% (v/v) glycerol. 


330 mM Tris-acetate (pH 7.9 at 37°C), 

100 mM Mg-acetate, 660 mM K-acetate, 

1% (v/v) Tween 20, 10 mM DTT. 


aphidicolin, N2-(p-n-butylphenyl) dGTP (BuPdGTP), 2-(p-n-butylanilino)-dATP 

(BuAdATP) (20). 

 

Note 

 

the phi29 reaction mixture may enhance 

DNA synthesis (8). 

Copy number of genome 

1.00E+08 

1.00E+07 

chromosome No 

6 8 15 18 total DNA 

Unbiased amplification of human genomic DNA 

with phi29 Thermo Scientific DNA Polymerase. 

100 copies (0.33 ng) of human genomic DNA were 

amplified with phi29 DNA Polymerase according 

to the procedure described by Dean, F.B., et al. (8). 

Radioactive [-33 1.00E+06 

1.00E+05 

1.00E+04 

1.00E+03 

1.00E+02 

1.00E+01 

1.00E+00 

tnf c-myc igf1r bcl2 

P]-dATP incorporation into the 

DE-81 adsorbable form was calculated to quantify 

amplification of total DNA. Amplification efficiency of 

specific genes located in different chromosomes was 

determined by real-time PCR using the ABI 7700. 

Use of this enzyme in certain applications may be covered by 

patents and may require a license. 


246 


#EP0041 500 u (10 u/μl) 



#EP0042 2500 u (10 u/μl) 




RNase H p.266 

DNase I, RNase-free p.255 



dNTP Mix, 10 mM each p.408 

Modified Nucleotides p.413 

0.5 M EDTA, pH 8.0 p.421 





» 6.3.1. Radioactive DNA labeling by 

nick-translation p.350 

» 6.3.2. Non-radioactive DNA labeling by 



Description 

DNA Polymerase I, a template-dependent DNA 

polymerase, catalyzes 5’3’ synthesis of DNA. 

The enzyme also exhibits 3’5’ exonuclease 

(proofreading) activity, 5’3’ exonuclease 

activity and ribonuclease H activity. 

Features 

Incorporates modified nucleotides 

(e.g., biotin-, digoxigenin-, aminoallyl-, 

fluorescently-labeled nucleotides). 


restriction enzymes, PCR and RT. 

Applications 

DNA labeling by nick-translation in 

conjunction with DNase I (1-3). 

Second-strand synthesis of cDNA in 

conjunction with RNase H (4). 

Concentration 

10 u/μl 

Source 

E.coli cells with a cloned polA gene from E.coli. 


103 kDa monomer. 


One unit of the enzyme catalyzes 

the incorporation of 10 nmol of 

deoxyribonucleotides into a polynucleotide 

fraction (adsorbed on DE-81) in 30 min at 

37°C, using poly(dA-dT)·poly(dA-dT) as a 

template·primer. 


mixture: 67 mM potassium phosphate (pH 7.4), 

6.7 mM MgCl2, 1 mM 2-mercaptoethanol, 

0.033 mM dATP, 0.033 mM dTTP, 

0.4 MBq/ml [ 3H]-dTTP and 

62.5 μg/ml poly(dA-dT)·poly(dA-dT). 



(pH 7.5), 0.1 mM EDTA, 1 mM DTT and 




100 mM MgCl2, 10 mM DTT. 


Inhibitors: metal chelators, PPi, Pi (at high 

concentrations) (5). 


by addition of EDTA. 

References 

1. Ausubel, F.M., et al., Current Protocols in Molecular 

 

York, 3.5.3-3.5.6., 1994-2005. 

2. 

Laboratory Manual, Third Edition, Cold Spring Harbor 

laboratory, Cold Spring Harbor, N. Y., 2001. 

3. Yu, H., et al., Cyanine dye dUTP analogs for enzymatic 

labeling of DNA probes, Nucleic Acids Res., 22, 

3226-3232, 1994. 

4. 

method for generating cDNA libraries, Gene, 25, 

263-269, 1983. 

5. Eun, H-M., Enzymology Primer for Recombinant DNA 

Technology, Academic Press, INC, 1996.

Klenow Fragment 

#EP0051 300 u (10 u/μl) 



#EP0052 1500 u (10 u/μl) 



#EP0054 LC, 300 u (2 u/μl) 






Terminal Deoxynucleotidyl Transferase (TdT) p.251 

RNase H p.266 



dNTP Mixes p.408 


0.5 M EDTA, pH 8.0 p.421 


References 


 

York, 3.5.7-3.5.10, 1994-2005. 

2. Feinberg, A.P., Vogelstein, B., A technique for radiolabeling 

DNA restriction endonucleases fragments to high specific 

activity, Anal. Biochem., 132, 6-13, 1983. 

3. Feinberg, A.P., Vogelstein, B., Addendum to: A technique 

for radiolabeling DNA restriction endonuclease fragments 

to high specific activity, Anal. Biochem., 137, 

266-267, 1984. 



3226-3232, 1994. 

5. Sanger, F., et al., DNA sequencing with chain-terminating 

inhibitors, Proc. Natl. Acad. Sci. USA, 74, 

5463-5467, 1977. 

6. Wallace, R.B., et al., Directed deletion of a yeast transfer 

RNA intervening sequence, Science, 209, 

1396-1400, 1980. 

7. Rougeon, F., et al., Insertion of rabbit -globin gene 

sequence into an E.coli plasmid, Nucleic Acids Res., 2, 

2365-2378, 1975. 


Technology, Academic Press, Inc., 1996. 

Description 

Klenow Fragment is the Large Fragment of DNA 

Polymerase I. It exhibits 5’3’ polymerase 

activity and 3’5’ exonuclease (proofreading) 

activity, but lacks 5’3’ exonuclease activity of 

DNA Polymerase I. 

Features 


(e.g., Cy3-, Cy5-, aminoallyl-, biotin-, 

digoxigenin- and fluorescently-labeled 

nucleotides). 


restriction enzymes, PCR, RT and T4 DNA 

Ligase. 

Applications 

DNA blunting by fill-in 5’-overhangs. (1). 

Random-primed DNA labeling (2-4). 

Labeling by fill-in 5’-overhangs of dsDNA. 

DNA sequencing by the Sanger method (5). 

Site-specific mutagenesis of DNA with 

synthetic oligonucleotides (6). 

Second strand synthesis of cDNA (7). 

Concentration 

10 u/μl 

2 u/μl, LC 

Source 

E.coli cells with a cloned fragment of the polA gene. 































» 3.2.2. Fill-in of 5’-overhangs with 


» 6.1.4. DNA 3’-end labeling by fill-in 

of 5’-overhangs p.349 


248 


#EP0421 300 u (5 u/μl) 



#EP0422 1500 u (5 u/μl) 








0.5 M EDTA, pH 8.0 p.421 





» 6.1.4. DNA 3’-end labeling by fill-in of 

5’-overhangs p.349 

» 6.2.1. Radioactive random-primed 

DNA labeling p.350 

» 6.2.2. Non-radioactive random-primed 

DNA labeling p.350 


Description 

Klenow Fragment, exo – is the Large Fragment 

of DNA Polymerase I. It exhibits 5’3’ 

polymerase activity, but lacks the 3’5’ 

and 5’3’ exonuclease activities of DNA 

Polymerase I. The 3’5’ exonuclease activity 

of the enzyme is eliminated by mutations in the 

3’5’-exonuclease active site (1). 

Features 

Lacks 3’5’ exonuclease activity. 

Incorporates modified nucleotides (e.g., Cy3-, 

Cy5-, fluorescein-, rhodamine-, aminoallyl-, 

biotin-labeled nucleotides). 


restriction enzymes, PCR and RT. 

Applications 

Random-primed DNA labeling (2-4). 


Strand displacement amplification (SDA) (5). 

DNA sequencing by the Sanger method (6). 

Concentration 

5 u/μl 

Source 

E.coli cells with a cloned DNA fragment of the 

mutated polA gene. 



























by the addition of EDTA. 

Note 

– , is not recommended 

for DNA blunting reactions prior to DNA 

ligation since it frequently adds one or more 

extra nucleotides to the 3’-terminus of 

blunt-end DNA substrates in a non-template 

directed fashion (8). 

References 

1. Derbyshire, V., et al., Genetic and crystallographic studies 

of the 3’,5’-exonucleolytic site of DNA polymerase I, 

Science, 240, 199-201, 1988. 


DNA restriction endonuclease fragments to high specific 


3. Feinberg, A.P., Vogelstein, B., Addendum to: A technique 

for radiolabeling DNA restriction endonuclease fragments 

to high specific activity, Anal. Biochem., 137, 

266-267, 1984. 



3226-3232, 1994. 

5. Walker, G.T., Empirical aspects of strand displacement 

amplification, PCR Methods Appl., 3, 1-6, 1993. 

6. Sanger, F., et al., DNA sequencing with chain-terminating 

inhibitors, Proc. Natl. Acad. Sci. USA, 74, 

5463-5467, 1977. 



8. 

catalyzed by DNA polymerase I of Escherichia coli,


#EP0061 100 u (5 u/μl) 



#EP0062 500 u (5 u/μl) 








0.5 M EDTA, pH 8.0 p.421 


References 

1. 

Laboratory Manual, Third edition, Cold Spring Harbor 



 

York, 3.5.11-3.5.12, 1994-2005. 

3. Challberg, M.D., Englund, P.T., Specific labeling of 

3’-termini with T4 DNA polymerase, Methods Enzymol., 

65, 39-43, 1980. 

4. Kunkel, I.A., et al., Rapid and efficient site-specific 

mutagenesis without phenotypic selection, Methods 

Enzymol., 154, 367-382, 1987. 

5. Haun, R.S., et al., Rapid, reliable ligation-independent 

cloning of PCR products using modified plasmid vectors, 


6. Wang, K., et al., A simple method using T4 DNA 

polymerase to clone polymerase chain reaction products, 




Description 

T4 DNA Polymerase, a template-dependent 

DNA polymerase, catalyzes 5’3’ synthesis 

from primed single-stranded DNA. The enzyme 

has a 3’5’ exonuclease activity, but lacks 

5’3’ exonuclease activity. 

Features 

Stronger 3’5’ exonuclease activity on 

single-stranded than on double-stranded 

DNA and greater (more than 200 times) than 

DNA polymerase I and Klenow fragment (1). 


restriction enzymes, PCR, RT and T4 DNA 

Ligase. 

Applications 

Blunting of DNA ends: fill-in 5’-overhangs or/ 

and removal of 3’-overhangs (1, 2). 

Blunting of PCR products with 3’-dA 

overhangs. 

Synthesis of labeled DNA probes by the 

replacement reaction (3). 

Oligonucleotide-directed site-specific 

mutagenesis (4). 

Ligation-independent cloning of PCR 

products (5, 6). 

Concentration 

5 u/μl 

Source 









fraction (adsorbed on DE-81) in 30 min at 37°C. 



6.7 mM MgCl2, 1 mM DTT, 16.7 mM (NH4) 2SO4, 0.2 mg/ml BSA, 0.033 mM of each dNTP, 

0.4 MBq/ml [ 3H]-dTTP and 0.2 mM 

heat-denatured and nuclease-digested calf 

thymus DNA. 


The enzyme is supplied in: 20 mM potassium 

phosphate (pH 7.5), 200 mM KCl, 2 mM DTT 

and 50% (v/v) glycerol. 



33 mM MgCl2, 5 mM DTT, 84 mM (NH4) 2SO4. Inhibition and Inactivation 

Inhibitors: metal chelators, nucleotide 

analogs 2(p-n-butylanilino)-dATP, 

N2-(p-n-butylphenyl)-dGTP), SH-blocking 

compounds (7). 

Inactivated by heating at 75°C for 10 min. 




» 3.2.1. Blunting of 5’- or 3’-overhangs 

with T4 DNA Polymerase p.295 


250 


#EP0081 300 u (10 u/μl) 






dNTP Mix, 10mM each p.408 


0.5 M EDTA, pH 8.0 p.421 






Description 

T7 DNA Polymerase, a template dependent DNA 

polymerase, catalyzes DNA synthesis in the 

5’3’ direction. It is a highly processive DNA 

polymerase allowing continuous synthesis of 

long stretches of DNA. The enzyme also exhibits 

a high 3’5’ exonuclease activity towards 

single- and double-stranded DNA. 

Features 

Strong 3’5’ exonuclease activity, 

approximately 1000-fold greater than Klenow 

Fragment (1). 


restriction enzymes. 

Applications 

Purification of covalently closed circular DNA 

by removal of residual genomic DNA. 

Primer extension reactions on long templates (1). 

DNA 3’-end labeling (1). 

Strand extensions in site-directed 


Fill-in blunting of 5’-overhang DNA. 

Second strand synthesis of cDNA (3). 

In situ detection of DNA fragmentation 

associated with apoptosis (4). 

Concentration 

10 u/μl 

Source 

Two E.coli strains, one with the cloned gene 5 of 

bacteriophage T7, and the other with the cloned 

trxA gene of E.coli. 


The T7 DNA Polymerase is composed of two 

subunits: an 80 kDa polypeptide (the product 

of gene 5 of bacteriophage T7) and a 12 kDa 

thioredoxin (from the trxA gene of E.coli). 





fraction (adsorbed on DE-81) in 30 min at 37°C. 



MgCl2, 1 mM DTT, 0.1 mg/ml BSA, 

0.33 mM of each dNTP, 0.4 MBq/ml [ 3H]-dTTP and 0.5 mM alkali-denatured calf thymus DNA. 



phosphate (pH 7.4), 1 mM DTT, 0.1 mM EDTA 






Inhibitors: metal chelators, modification 

reagents (acetic anhydride, N-ethylmaleimide 

inactivate the 3’5’ exonuclease activity but 

not the polymerase activity) (5). 


Note 

 

times (6). 

References 

1. 

Laboratory Manual, Third edition, Cold Spring Harbor 


2. Bebenek, K., Kunkel, T.A., The use of native T7 DNA 

polymerase for site-directed mutagenesis, Nucleic Acids 

Res., 17, 5408, 1989. 

3. Bodescot, M., Brison, O., Efficient second-strand cDNA 

synthesis using T7 DNA polymerase, DNA and Cell Biology, 

13, 977-985, 1994. 

4. Wood, K.A., et al., In situ labeling of granule cells for 

apoptosis-associated DNA fragmentation reveals different 

mechanisms of cell loss in developing cerebellum, Neuron, 

11, 621-632, 1993. 



6. Bodescot, M., Brison, O., T7 DNA polymerase requires 

unusual reaction conditions for blunt-ending activity, Anal. 

Biochemistry, 216, 234-235, 1994.

Terminal Deoxynucleotidyl Transferase (TdT) 

#EP0161 500 u (20 u/μl) 



#EP0162 2500 u (20 u/μl) 




Uracil-DNA Glycosylase (UDG, UNG) p.273 

Endonuclease V, T.maritima (Endo V) p.257 

Biotin Chomogenic Detection Kit p.345 


dNTPs p.408 


0.5 M EDTA, pH 8.0 p.421 


References 

1. 

Enzymes, the third edition (Boyer, P.D., ed.), Academic Press, 

New York, vol.10, 145-171, 1974. 

2. Deng, G.R., Wu, R., Terminal transferase: Use in the tailing 

of DNA and for in vitro mutagenesis, Meth. Enzymol., 100, 

96-116, 1983. 

3. Eschenfeldt, W.H., et al., Homopolymeric tailing, Meth. 

Enzymol., 152, 337-342, 1987. 

4. Tu, C.-P.D., Cohen, S.N., 3’-end labeling of DNA with 

[- 32 P]-cordycepin-5’-triphosphate, Gene, 10, 177-183, 1980. 

5. Vincent, C., et al., Synthesis of 8-(2,4-dinitrophenyl-2,6aminohexyl)aminoadenosine-5’-triphosphate: 

Biological 

properties and potential uses, Nucleic Acids Res., 

10, 6787-6796, 1982. 

6. Kumar, A., et al., Nonradioactive labeling of synthetic 

oligonucleotide probes with terminal deoxynucleotidyl 

transferase, Anal. Biochem., 169, 376-382, 1988. 

7. Gaastra, W., Klemm, P., Radiolabeling of DNA with terminal 

transferase, Methods in Molecular Biology, vol.2: Nucleic Acids 

 

8. Igloi, G.L., Schiefermayr, E., Enzymatic addition of fluorescein- 

or biotin-riboUTP to oligonucleotides results in primers suitable 

for DNA sequencing and PCR, BioTechniques, 15, 

486-497, 1993. 

9. Frohman, M.A., et al., Rapid Production of Full-Length cDNAs 

from Rare Transcripts: Amplification Using a Single Gene- 

Specific Oligonucleotide Primer, Proc. Natl. Acad. Sci. USA, 85, 

8998-9002, 1988. 

10. Gorczyca, W., et al., Detection of DNA strand breaks 

in individual apoptotic cells by the in situ terminal 

deoxynucleotidyl transferase and nick translation assays, 

Cancer Res., 53, 1945-1951,1993. 

Description 

Terminal Deoxynucleotidyl Transferase 

(TdT), a template-independent DNA 

polymerase, catalyzes the repetitive addition 

of deoxyribonucleotides to the 3’-OH of 

oligodeoxyribonucleotides and single- or 

double-stranded DNA (1). TdT requires an 

oligonucleotide of at least three nucleotides 

to serve as a primer. With an RNA template, 

TdT shows variable performance which 

strongly depends upon the tertiary structure 

of the acceptor RNA 3’-end and the nature of 

nucleotide. Generally, the polymerase activity 

with an RNA template is lower compared to the 

activity with a DNA template. 

Features 


(e.g., fluorescein-, biotin-, aminoallyl-labeled 

nucleotides). 

Applications 

Production of synthetic homo- and 

heteropolymers (1). 

Homopolymeric tailing of linear duplex DNA 

with any type of 3’-OH terminus (2, 3), 

see protocol below. 

Oligodeoxyribonucleotide and DNA labeling 

(2, 4-8). 

5’-RACE (Rapid Amplification of cDNA Ends) (9). 

In situ localization of apoptosis (10). 

Concentration 

20 u/μl 

Source 

E.coli cells carrying a cloned gene encoding calf 

thymus terminal deoxynucleotidyl transferase. 



incorporation of 1 nmol of deoxythymidylate into 

a polynucleotide fraction (adsorbed on DE-81) in 

60 min at 37°C. 


mixture: 200 mM potassium cacodylate 

(pH 7.2), 

1 mM CoCl2, 0.01% (v/v) Triton X-100, 

10 μM oligo(dT) 10, 1 mM dTTP and 

0.4 MBq/ml [ 3H]-dTTP. Storage Buffer 


acetate (pH 6.8), 2 mM 2-mercaptoethanol, 

0.01% (v/v) Triton X-100 and 50% (v/v) glycerol. 


1 M potassium cacodylate, 125 mM Tris, 

0.05% (v/v) Triton X-100, 

5 mM CoCl2 (pH 7.2 at 25°C). 


Inhibitors: metal chelators, ammonium, 

chloride, iodide, phosphate ions. 



Protocol for tailing of DNA 3’-termini 

1. Prepare the following reaction mixture: 

5X reaction buffer for Terminal Deoxynucleotidyl Transferase 4 μl 

DNA fragments 1 pmol of 3’-ends 

(dATP or dTTP) or 

(dGTP or dCTP) 

Note 

 

Reaction Buffer is incompatible with 

downstream applications. It is necessary to 

remove CoCl2 from the reaction mixture by 

spin column or phenol/chloroform extraction 

and subsequent ethanol precipitation. 

130 pmol or 

60 pmol 

Terminal Deoxynucleotidyl Transferase (#EP0161) 1.5 μl (30u) 

Water, nuclease-free (#R0581) to 20 μl 


2. Incubate the mixture at 37°C for 15 min. 

3. Stop the reaction by heating at 70°C for 10 min or by the addition of 2 μl 0.5 M EDTA (#R1021). 

Note 

 

end of DNA. 

 

3’-overhangs are tailed with higher efficiency than recessed or blunt ends. 


» 6.1.5. DNA and oligonucleotide 3’-end 

labeling by tailing p.349 


252 

Terminal Transferase 

Formerly manufactured by Finnzymes OY, now part of Thermo Fisher Scientific 

#F-203S 500 u (15 u/μl) 


10x TdT Buffer 1.5 ml 

25 mM CoCl2 1.5 ml 

#F-203L 


2500 u (15 u/μl) 

10x TdT Buffer 1.5 ml 

25 mM CoCl2 1.5 ml 



Endonuclease V, T.maritima (Endo V) p.257 

Biotin Chomogenic Detection Kit p.345 


Deoxyribonucleoside Triphosphates p.406 


0.5 M EDTA, pH 8.0 p.421 



Description 

Terminal transferase (TdT) is a template 

independent polymerase that catalyzes the 

addition of deoxynucleotides to the 3´ hydroxyl 

terminus of DNA molecules. Protruding, 

recessed or blunt ended double or single 

stranded DNA molecules serve as 

a substrate for TdT. 

Applications 

Addition of homopolymeric tails to plasmid 

DNA and to cDNA. 

Double- or single-stranded DNA 3´-termini 

labeling with radioactively labeled or nonradioactively 

labeled nucleotides. 

Addition of single nucleotides to the 3´ ends 

of DNA for in vitro mutagenesis. 

Production of synthetic homo- and 

heteropolymers. 

RACE (Rapid Amplification of cDNA Ends). 

Concentration 

15 u/μl 

Source 

E.coli cells with a cloned gene of terminal 

transferase from calf thymus. 



incorporation of 1 nmol of deoxythymidylate into 

a polynucleotide fraction (adsorbed on DE-81) in 

60 min at 37°C. 


mixture: 200 mM potassium cacodylate 

(pH 7.2), 

1 mM CoCl2, 0.01% (v/v) Triton X-100, 

10 μM oligo(dT) 10, 1 mM dTTP and 

0.4 MBq/ml [ 3H]-dTTP. Storage Buffer 

Enzyme is supplied in: 60 mM KPO4, 150 

mM KCl, 1 mM -mercaptoethanol, 1% Triton 

X-100, 50% glycerol (v/v), (pH 7.2 at 25°C). 

10x TdT Buffer 


500 mM Potassium acetate. 

Recommended concentration 

of CoCl2 in 1X reaction buffer is 1.5 mM. 

Note 

2 the TdT 

Reaction Buffer is incompatible with 

downstream applications. It is necessary to 

remove CoCl 2 from the reaction mixture by 

spin column or phenol/chloroform extraction 

and subsequent ethanol precipitation. 

 

with 10X reaction buffer. This product is 

recommended for use in small volume 

reactions and/or with dilute samples. 

Find Thermo Scientific thermostable DNA polymerases, reverse transcriptases, 

qPCR reagents, PCR & qPCR instruments and consumables at 

www.thermoscientific.com/pcr

Bsm DNA Polymerase, Large Fragment 

#EP0691 1600 u (8 u/μl) 


10X Bsm Buffer 1.25 ml 



dNTP Mix, 10mM each p.408 


dUTP p.409 

Biotin-11-dUTP p.414 


References 

1. Tsugunori Notomi, et al., Loop-mediated isothermal 

amplification of DNA, Nucleic Acids Res., v. 28, 

No. 12, e63, 2000. 

2. Masaki Imai, et al., Rapid diagnosis of H5N1 avian 

influenza virus infection by newly developed influenza 

H5 hemagglutinin gene-specific loop-mediated isothermal 

 

141, 173-180, 2007. 

3. Dean, F.B., et al., Comprehensive human genome 

amplification using multiple displacement amplification, 

Proc. NatI. Acad. Sci. USA, 99, 5261-5266, 2002. 

4. 

real-time isothermal DNA amplification, Nucleic Acids 

Res., v.34, No.11, e81, 2006. 

Description 

Bsm DNA Polymerase is the large fragment 

portion of the Bacillus smithii DNA polymerase 

enzyme, which catalyzes 5’3’ synthesis of 

DNA and lacks 5’3’ and 3’5’ exonuclease 

activities. The enzyme has strong strand 

displacement activity and is active in wide 

range of temperatures from 30°C to 63°C, 

with an optimum of activity at 60°C. Bsm 

DNA Polymerase is an enzyme with high 

functional similarity to Bst DNA Polymerase, 

Large fragment and can replace Bst in most 

applications. The enzyme is not suitable for use 

in PCR. 

Features 

Thermophilic DNA polymerase with strong 

strand displacement activity. 

Applications 

Isothermal DNA amplification by the method of: 

– loop- mediated isothermal amplification, 

(LAMP) (1, 2), 

– whole genome amplification (WGA) (3), 

– ramification amplification (RAM) (4). 

Random- primed DNA labeling. 


Concentration 

8 u/μl 



Source 

E.coli cells with a cloned part of polA gene from 

Bacillus smithii. 







fraction (absorbed on DE-81) in 30 min at 60°C. 


mixture 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 100 μg/ml BSA, 0.75 mM activated salmon 

milt DNA, 0.2 mM of each dNTP, 0.4 MBq/ml 

[H3 ]-dTTP. 


Enzyme is supplied in: 



50% glycerol. 

10X Bsm Buffer 


100 mM KCl, 100 mM (NH4) 2SO4, 20 mM MgSO4, 1% Tween 20. 

Inactivation 

By heating at 80°C for 10 min. 




» 6.2.1. Radioactive random-primed DNA 

labeling p.350 


254 

Deoxyribonucleases (DNases) 

Table 2.4. Properties of deoxyribonucleases (DNases). 

Nuclease Applications Substrate Type 

DNase I, 

RNase-free 

Endonuclease IV, 

E.coli (Endo IV) 

Endonuclease V, 

T.maritima 

(Endo V) 

Exonuclease I 

(Exo I) 

Exonuclease III 

(Exo III) 

Lambda 

Exonuclease 


 

 

following in vitro transcription. 

ssDNA 

 

to RT-PCR. 

 

 

 

randomly overlapping DNA inserts. 

a 

dsDNA 

DNA in RNA-DNA 

hybrids 

Studies of DNA damage and repair. 

Single cell electrophoresis. 

Antitumor drug research. 

DNA structure research. 

SNP analysis. 

AP DNA 

Specificity, 

polarity of 

cleavage 

Endonuclease b Sequence and 

base unspecific 

AP endonuclease 

c 

5’ to an abasic 

site, 3’5’ 

exonuclease 

Reaction 

products 

5’-dNMPs, 

5’-oligonucleotides 

DNA with 

nicks 5’ to 

AP sites 


Heating at 

65°C for 10 min in the presence 

of EGTA or EDTA 

EN0521/3/5 255 

Heating at 80°C for 

15 min d EN0591 256 

 

mutation research. 

Second 

 

repair. 

Deaminated DNA 

phosphodiester 

Endonuclease 

Nicked DNA 

bond 3’ to the 

 

 

lesion 

e Heating at >95°C for 10 min 

in the presence of EDTA 

 

 

acid mixtures. 

Assay for the presence of singlestranded 

DNA with a 3’-hydroxyl 

terminus. 

 

deletions in DNA fragments. 

 

sequencing. 

 

 

 

probes. 

ssDNA f Exonuclease g 3’5’ 

dsDNA (with 

nicks, blunt ends, 

5’-overhangs) h 

AP DNA 

RNA in 

RNA-DNA hybrids 

DNA with 3’-phosphorylated 

ends 

Exonuclease i 3’5’ 

AP endonuclease 

RNase H 

(exonuclease) 

5’ to an abasic 

site 

3’-phosphatase – 

5’-dNMPs, 

5’-terminal 

dinucleotides 

5’-dNMPs, 

ssDNA 

DNA with 

nicks 5’ to 

AP sites 

3’5’ NMPs 

3’-OH ends 

of DNA 

Generation of ssPCR products for 

sequencing, SSCP analysis, rolling 

circle amplification. 

Producing ssDNA from 

dsDNA fragments. 

Cloning of PCR products. 


dsDNAj Exonuclease 5’3’ 5’-dNMPs 

a – ssDNA is cleaved slower than dsDNA. 

b – in the presence of Mg 2+ , the resulting DNA fragments have overhang termini; in the presence of Mn 2+ the individual fragments are shorter, 

they are blunt-end or have 1-2 nucleotide long overhang termini. 

c – also removes 3’-blocking groups (e.g., 3’-phosphoglycolate and 3’-phosphate) from damaged ends of dsDNA. 

d – the enzyme has no requirement for Mg 2+ and is active in the presence of EDTA. 

e – when enzyme is in excess, complementary strand is also nicked, generating a double-stranded break. 

f – does not cleave DNA chains with terminal 3’-phosphoryl or acetyl groups. Not suitable for removing 3’-overhang ends of dsDNA – this activity is greatly reduced. 

g – also exhibits DNA deoxyribophosphodiesterase activity. 

h – not active on 3’-overhang ends of DNA that are at least 4 base long or on phosphorothioate-linked nucleotides or ssDNA. 

i – it is also a DNA 3’-phosphomonoesterase and 3’-repair diesterase. 

j – selectively digests the 5’-phosphorylated strand of dsDNA. Exhibits greatly reduced activity on ssDNA and non-phosphorylated DNA; 

has limited activity at gaps in DNA and no activity at nicks. 

ss – single-stranded. 


dNMP – deoxyribonucleoside monophosphate. 

NMP – ribonucleoside monophosphate. 

AP – apurinic/apyrimidinic. 

SSCP – single-stranded conformation polymorphism. 

Heating at 

80°C for 15 min 

Heating at 


Heating at 


EN0141 257 

EN0581/2 258 

EN0191 259 

EN0561/2 260

DNase I, RNase-free 

#EN0521 1000 u (1 u/μl) 

#EN0523 HC,1000 u (50 u/μl) 


10X Reaction Buffer with MgCl 2 1 ml 

50 mM EDTA 1 ml 

#EN0525 1000 u (1 u/μl) 


10X Reaction Buffer with MgCl2 1 ml 

10X Reaction Buffer w/o MnCl2 1 ml 

100 mM MnCl2 1 ml 


#B43 10X Reaction Buffer with 

MgCl 2 for DNase I, 

RNase-free 1 ml 




DNA Polymerase I p.246 

dNTP Sets p.408 


0.5 M EDTA, pH 8.0 p.421 


 

 

References 

1. 

A Laboratory Manual, the third edition, Cold Spring Harbor 


2. Kienzle, N., et al., DNase I treatment is a prerequisite for 

the amplification of cDNA from episomal-based genes, 


3. Anderson, S., Shotgun DNA sequencing using cloned 

DNaseI-generated fragments, Nucleic Acids Res., 9, 

3015-3027, 1981. 

4. 

5. Wiame, I., et al., Irreversible heat inactivation of DNase 

I without RNA degradation, BioTechniques, 29, 

252-256, 2000. 

Description 

DNase I iis an endonuclease that digests 

single- and double-stranded DNA. The enzyme 

hydrolyzes phosphodiester bonds producing 

mono- and oligodeoxyribonucleotides with 

5’-phosphate and 3’-OH groups. 

The enzyme activity is strictly dependent on Ca2+ and is activated by Mg2+ or Mn2+ ions 

2+ , DNase I cleaves 

each strand of dsDNA independently, in a 

statistically random fashion (1); 

2+ , the enzyme cleaves 

both DNA strands at approximately the same 

site, producing DNA fragments with bluntends 

or with overhang termini of only one or 

two nucleotides (1). 

Features 

Recombinant enzyme. 

Purified from non-animal host with a lower 

level of intrinsic RNases. 

Applications 

Preparation of DNA-free RNA (1). 

Removal of template DNA following in vitro 

transcription (1). 

Preparation of DNA-free RNA prior to 

RT-PCR (2). 

DNA labeling by nick-translation in 

conjunction with DNA Polymerase I (1). 

Studies of DNA-protein interactions by 

DNase I, RNase-free footprinting (1). 

Generation of a library of randomly 

overlapping DNA inserts. Reaction buffer 

containing Mn2+ is used (3). 

5’ 

3’ 

dsDNA 

P 

P – phosphate 

3’ 

5’ 

P P P 

5’ 3’ 

5’ 

3’ 

5’ 

3’ 

dsDNA 

3’ OH 

5’ P 

P 5’ 

OH 3’ 

P 

Mg 2+ 

DNase I activity in the presence of Mg2+ or Mn2+ P 5’ 3’ OH P 5’ 3’ 

OH 3’ 


5’ P OH 3’ 5’ 

. 

P 

Mn 2+ 

3’ 

5’ 

3’ OH 

5’ P 

Concentration 

1 u/μl 

50 u/μl, HC 

Source 

E.coli cells with a cloned gene encoding bovine 

DNase I. 




One unit of the enzyme completely degrades 

1 μg of plasmid DNA in 10 min at 37°C. 



MgSO4, 1 mM CaCl2, 1 μg of pUC19 DNA. One 

DNase I unit is equivalent to 0,3 Kunitz unit (4). 



(pH 7.5), 10 mM CaCl2 and 50% (v/v) glycerol. 

10X Reaction Buffer with MgCl2 100 mM Tris-HCl (pH 7.5 at 25°C), 

25 mM MgCl2, 1 mM CaCl2. 10X Reaction Buffer without MnCl2 100 mM Tris-HCl (pH 7.5 at 25°C), 1 mM CaCl2. Recommended concentration of MnCl2 in 

1X reaction buffer is 10 mM. 


 

(e.g., Zn) in millimolar concentrations, 

SDS (even at concentrations less than 

0.1%), reducing agents (DTT and 

-mercaptoethanol), ionic strength above 

50-100 mM. 

 

the presence of EGTA or EDTA (use at least 1 

mol of EGTA/EDTA per 1 mol of Mn2+ /Mg2+ (5)). 

Note 

 

Mix gently by inverting the tube. Do not 

vortex. 


» 4.6. Removal of template DNA after in vitro 

transcription p.323 

» 4.7. Removal of genomic DNA from RNA 

preparations p.323 

» 6.3.1. Radioactive DNA labeling by 





256 

Endonuclease IV, E.coli (Endo IV) 

#EN0591 100 u (2 u/μl) 





Exonuclease I (Exo I) p.258 



Description 

Endonuclease IV (Endo IV) recognizes apurinic/ 

apyrimidinic (AP) sites of dsDNA and cleaves 

the phosphodiester bond 5’ to the lesion 

generating a hydroxyl group at the 3’-terminus. 

The enzyme can also act as a 3’-diesterase that 

is able to release 3’-phosphoglycolate or 

3’-phosphate from the damaged ends of dsDNA (1). 

Endo IV also demonstrates 3’5’ exonuclease 

activity. The rate of enzyme activity is 

sensitive to ionic strength, metal ions, EDTA, 

and reducing conditions. Substrates with 

3’-recessed ends are preferred substrates for 

the 3’5’ exonuclease activity (2). 

The enzyme has no requirement for Mg 2+ but is 

more active in the presence of Mg 2+ . 

Applications 

Studies of DNA damage and repair (3, 4, 5). 

Single cell electrophoresis (comet assay) (6). 

Antitumor drug research (4). 

DNA structure research (5, 7). 

SNP analysis (8). 

Concentration 

2 u/μl 

5’ 

3’ 

dsDNA 

5’ 

3’ 


a – abasic site 

P-a-P 

OH 

Endonuclease IV activity. 

P-a-P 

3’ 

5’ 

3’ 

5’ 

Source 

E.coli cells with a cloned nfo gene. 




One unit of the enzyme relaxes 1 μg of depurinated, 

supercoiled plasmid DNA in 30 min at 37°C. 


mixture: 50 mM Tris-acetate (pH 7.5), 

50 mM KCl, 1 mM EDTA, 0.05% (v/v) Triton 

X-100 and 

2 μg of partially depurinated pUC19 DNA. 



50 mM Tris-acetate (pH 7.7), 50 mM KCl, 

1 mM DTT, 0.05% (v/v) Triton X-100 and 



500 mM Tris-acetate (pH 7.5), 500 mM KCl, 

10 mM EDTA, 0.5% (v/v) Triton X-100. 


 

EDTA during the reaction, but becomes 

sensitive to even submillimolar quantities of 

chelators when no DNA substrate is present. 

 

References 

1. Escherichia coli endonuclease 

 

2. Kerins, S.M., et al., Characterization of an endonuclease IV 

 

3048-3054, 2003. 

3. Demple, B., Harrison, L., Repair of oxidative damage to 

DNA: Enzymology and biology, Annu. Rev. Biochem., 63, 

915-948, 1994. 

4. In vitro detection of endonuclease 

IV – specific DNA damage formed by bleomycin in vivo, 


5. 

formamidopyrimidine lesions Fapy.dA and Fapy.dG in DNA 

using endonuclease IV, Biochemistry, 43, 

13397-13403, 2004. 

6. Collins, A.R., The comet assay for DNA damage and repair, 

Molec. Biotechnol., 26, 249-261, 2004. 

7. 

endonuclease IV and its DNA complex: double-nucleotide 

flipping at abasic sites and three-metal-ion catalysis, Cell, 

98, 397-408, 1999. 

8. Kutyavin I.V., et al., A novel endonuclease IV post-PCR 

genotyping system, Nucleic Acids Res., 29, 1-9, 2006.

Endonuclease V, T.maritima (Endo V) 

#EN0141 250 u (5 u/μl) 




dNTP Sets and Mixes p.408 

dUTP p.409 

dITP p.410 


References 

1. 

endonuclease V from Thermotoga maritima: recognition 

and strand nicking mechanism, Biochemistry, 40, 

8738-8748, 2001. 

2. Hitchcock, T.M., et al., Cleavage of deoxyoxanosinecontaining 

oligodeoxyribonucleotides by bacterial 

endonuclease V, Nucleic Acids Res., 32, 

4071-4080, 2004. 

3. Pincas, H., et al., High sensitivity EndoV mutation scanning 

through real-time ligase proofreading, Nucleic Acids Res, 

32, 148, 2004. 

4. 

scanning method especially suited for analysis of 

neoplastic tissue, Oncogene, 21, 1909-1921, 2002. 

Description 

Endonuclease V, T.maritima (Endo V), is a 

3’-endonuclease involved in DNA repair which 

initiates removal of deaminated bases from 

damaged DNA, including uracil, hypoxanthine 

and xanthine. 

Endonuclease V is also active toward abasic 

sites and urea sites, base pair mismatches, flap 

and pseudo Y structures, and small insertions/ 

deletions in DNA molecules. The cleavage site 

generated by Endonuclease V is at the second 

phosphodiester bond 3’ to a lesion. When 

the enzyme is in excess, the primary nicked 

products experience a second nicking event on 

the complementary strand, leading to a doublestranded 

break. At low concentrations, however, 

Endonuclease V first nicks a DNA strand at the 

lesions located closer to the 5’-end of DNA 

molecule. Single-stranded DNA is cleaved with 

much lower efficiency. Mg 2+ or Mn 2+ ions are 

required for enzyme activity (1, 2, 3). 

Features 

Temperature optimum of activity at 65-70°C. 

Applications 

High-throughput methods for mutation 

research (3, 4). 

Studies in mutagenesis and DNA repair. 

Mismatch cleavage. 

Genotyping. 

5’ 

3’ 

dsDNA 

5’ 

3’ 


X – nucleoside with deaminated base, i.e. uracil, 

hypoxanthine, xantine; abasic site, urea site or 

mismatched nucleoside (A=G>T>C) 

N – nucleoside, adjacent to improper nucleoside 

Endonuclease V activity. 

P-X-P-N-P 

P-X-P-N-3’ 

5’ P 

3’ 

5’ 

3’ 

5’ 



Concentration 

5 u/μl 

Source 

E.coli with a cloned nfi gene of Thermotoga 

maritima. 




One unit of the enzyme relaxes one μg of 

depurinated, supercoiled plasmid DNA in 

30 min at 65°C. Enzyme activity is assayed in 

the following mixture: 25 mM HEPES-NaOH 

(pH 7.4), 5 mM MgCl2, 5 mM DTT, 2% (v/v) 

glycerol, 2 μg of depurinated pUC19 DNA. 


The enzyme is supplied in: 20 mM HEPES-NaOH 

(pH 7.4), 5 mM DTT, 50 mM NaCl, 



250 mM HEPES-NaOH (pH 7.4 at 25°C), 

50 mM MgCl2, 50 mM DTT, 20% glycerol. 


 

described. 

Inactivated by heating in boiling water 

bath for 10 min, preferably in the 

presence of EDTA. 


258 

Exonuclease I (Exo I) 

#EN0581 4000 u (20 u/μl) 



#EN0582 20,000 u (20 u/μl) 






Endonuclease IV, E.coli (Endo IV) p.256 





» 4.8. PCR product clean-up prior to 

sequencing p.323 


Description 

Exonuclease I (ExoI) degrades singlestranded 

DNA in a 3’5’ direction, releasing 

deoxyribonucleoside 5’-monophosphates in 

a stepwise manner and leaving 5’-terminal 

dinucleotides intact. It does not cleave DNA 

strands with terminal 3’-OH groups blocked by 

phosphoryl or acetyl groups (1). 

Features 

Active in Thermo Scientific buffers for PCR. 

Applications 

Primer removal from PCR mixtures: 

– prior to PCR product sequencing (2), 

– for one-tube “megaprimer” PCR 


Removal of single-stranded DNA containing a 

3’-hydroxyl terminus from nucleic acid mixtures. 

Assay for the presence of single-stranded 

DNA with a 3’-hydroxyl terminus (4). 

Concentration 

20 u/μl 

Source 

E.coli cells with a cloned E.coli sbcB gene. 

5’ 3’ OH 

sDNA or 

oligonucleotide Mg2+ 5’ 

5’ NpN-OH 3’+ 5’ dNMPs 

Exonuclease I activity. 

3’ OH + 5’ dNMPs 




One unit of the enzyme catalyzes the release of 

10 nmol of acid soluble nucleotides in 30 min 

at 37°C. 


mixture: 67 mM glycine-KOH (pH 9.5), 

6.7 mM MgCl 2, 1 mM DTT and 

0.17 mg/ml single-stranded [ 3 H]-DNA. 



20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 

1 mM DTT and 50% (v/v) glycerol. 


670 mM glycine-KOH (pH 9.5 at 25°C), 



 


Note 

 

3’-overhangs in dsDNA. 

References 

1. Lehman, I.R., Nussbaum, A.L., The deoxyribonucleases of 

Escherichia coli. V. On the specificity of exonuclease 

 

2628-2636, 1964. 

2. Werle, E., et al., Convenient single-step, one tube 

purification of PCR products for direct sequencing, Nucleic 

Acids Res., 22, 4354-4355, 1994. 

3. Nabavi S., Nazar R.N., Simplified one-tube “megaprimer” 

polymerase chain reaction mutagenesis, Anal Biochem., 2, 

346-348, 2005. 

4. 

enzyme of Escherichia coli K-12 by Ca 2+ 

254, 8646-8652, 1979. 

5. Sasaki, Y., Miyoshi, D. and Sugimoto, N., Regulation of 

DNA nucleases by molecular crowding., Nucleic Acids 

Res., 35, 4086-4093, 2007.

Exonuclease III (Exo III) 

#EN0191 4000 u (200 u/μl) 




S1 Nuclease p.269 


Glycogen p.423 


References 

1. Rogers, S.G., Weiss, B., Exonuclease III of Escherichia 

coli K-12, an AP endonuclease, Methods Enzymol., 65, 

201-211, 1980. 

2. Escherichia coli 

Exonuclease III, Anal. Biochem., 209, 238-246, 1993. 

3. Henikoff, S., Unidirectional digestion with exonuclease III 

creates targeted breakpoints for DNA sequencing, Gene, 

28, 351-359, 1984. 

4. Guo, Li-He., Wu, R., New rapid methods for DNA 

sequencing based on exonuclease III digestion 

followed by repair synthesis, Nucleic Acids Res., 10, 

2065-2084, 1982. 

5. Vandeyar, M.A., et al., A simple and rapid method for the 

selection of oligodeoxynucleotide-directed mutants, Gene, 

65, 129-133, 1988. 

6. Li, Ch., Evans, R.M., Ligation independent cloning 

irrespective of restriction site compatibility, Nucleic Acids 

Res., 25, 4165-4166, 1997. 

7. Eun, H.-M. Enzymology Primer for Recombinant DNA 

Technology, Academic Press. Inc., 1996. 

8. 

of exonuclease III by varying the sodium chloride 

concentration, BioTechniques, 7, 932-933, 1989. 

Description 

Exonuclease III (Exo III) exhibits four different 

catalytic activities (1): 

5’ exodeoxyribonuclease activity specific 

for double-stranded DNA: 

Exo III degrades dsDNA from blunt 

ends, 5’-overhangs or nicks, releasing 

5’-mononucleotides from the 3’-ends of DNA 

strands and producing stretches of singlestranded 

DNA. It is not active on 3’-overhang 

ends of DNA that are at least four bases long 

and do not carry a 3’-terminal C-residue (2); on 

single-stranded DNA, or on phosphorothioatelinked 

nucleotides. 

 

Exo III removes the 3’-terminal phosphate 

and generates a 3’-OH group. 

 

Exo III degrades the RNA strand in RNA-DNA 

hybrids. 

 

Exo III cleaves phosphodiester bonds at 

apurinic or apyrimidinic sites to produce 

5’-termini that are base-free deoxyribose 

5’-phosphate residues. 

Features 


Restriction Enzymes. 

Applications 

Creation of unidirectional deletions in 

DNA fragments in conjunction with S1 

Nuclease (2, 3). 

Generation of a single-stranded template 

for dideoxy-sequencing of DNA (4). 

Site-directed mutagenesis (5). 

Cloning of PCR products (6). 

Preparation of strand-specific probes. 

5’ 

3’ 

dsDNA 

Exonuclease III activity. 

Mg 2+ 

3’ 

5’ 

5’ 3’ 

3’ 5’ 

+ 

5’ dNMPs 

Concentration 

200 u/μl 

Source 

E.coli cells with a cloned E.coli xth gene. 





1 nmol of acid soluble reaction products from 

E.coli [ 3H]-DNA in 30 min at 37°C. 


mixture: 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM DTT and 0.05 mM sonicated E.coli 

[ 3H]-DNA. Storage Buffer 



1 mM DTT and 50% (v/v) glycerol. 



6.6 mM MgCl2. Inhibition and Inactivation 

 

benzoate (50-90% inhibitory at 0.1 mM) (7). 

 

Note 

 

upon temperature, salt concentration 

and the molar ratio of DNA to enzyme 

in the reaction mixture (4, 8). Optimal 

reaction conditions should be determined 

experimentally. 





260 

Lambda Exonuclease 

#EN0561 1000 u (10 u/μl) 



#EN0562 5000 u (10 u/μl) 




T4 Polynucleotide Kinase (T4 PNK) 

FastAP Thernosensitive Alkaline 

p.243 








Description 

Lambda Exonuclease is a highly processive 

5’3’ exodeoxyribonuclease. It selectively 

digests the 5’-phosphorylated strand of 

double-stranded DNA. The enzyme exhibits 

low activity on single-stranded DNA and nonphosphorylated 

DNA, has no activity at nicks 

and limited activity at gaps in DNA (1, 2). 

Features 

Active in Thermo Scientific buffers for PCR. 

Applications 

Generating single-stranded PCR products for 

use in: 

– DNA sequencing (3), 

– analysis of DNA single-strand 

conformation polymorphism (SSCP) (4), 

– rolling circle amplification. 

Producing single-stranded DNA from doublestranded 

DNA fragments. 


Concentration 

10 u/μl 

Source 

E.coli cells with a cloned exo gene of phage 

lambda. 

HO 5’ 

HO 3’ 

dsDNA 

Mg 2+ 

3’ 

5’ 

OH 

P 

HO 5’ 3’ OH 

+ 

5’ dNMPs 


Lambda Exonuclease activity. 







10 nmol of acid soluble reaction products from 

double-stranded substrate in 30 min at 37ºC. 


mixture: 67 mM glycine-KOH (pH 9.4), 

2.5 mM MgCl2, 0.1% (v/v) Triton X-100 and 

20 μg/ml sonicated E.coli [³H]-DNA. 




1 mM DTT, 50 mM NaCl, 0.1% (v/v) Triton X-100 



670 mM glycine-KOH (pH 9.4), 25 mM MgCl2, 0.1% (v/v) Triton X-100. 


 

concentration (0.2 M KCl, 0.1 M NaCl), 

p-chloromercuribenzoate. 

 

References 

1. : 

 

679-686, 1967. 

2. 

interaction of lambda exonuclease with the ends of DNA, 


3. Higuchi, R.G., Ochman, H., Production of single-stranded 

DNA templates by exonuclease digestion following the 

polymerase chain reaction, Nucleic Acids Res., 17, 

5865, 1989. 

4. Schwieger, F., Tebbe, C.C., A new approach to utilize PCRsingle-stranded-conformation 

polymorphism for 16S rRNA 

gene-based microbial community analysis, Appl. Environ. 

Microbiol. 64, 4870-4876, 1998. 

5. Tseng, H., DNA cloning without restriction enzyme and 

ligase, Biotechniques. 27, 1240-1244, 1999.

Ribonucleases (RNases) 

Table 2.5. Properties of ribonucleases (RNases). 

Ribonuclease Applications 

RNase A, DNase and 

protease-free 

RNase T1 

RNase A/T1 Mix 

RNase I 

RNase H 

PowerCut Dicer 




 

DNA preparations. 

 

preparations. 

 

 

DNA or RNA. 

 

 

 

 

preparations. 

 

transcripts synthesized in vitro from DNA 

templates containing a “G-less cassette”. 

 

 

preparations. 

 

 

 

preparations. 

 

 

second strand cDNA. 

 

synthesis. 

 

of mRNA after the hybridization with 

oligo(dT). 

 

in vitro polyadenylation reaction 

products. 

 

studies. 

Reaction 

catalyzed 

Cleavage of 

phosphodiester bonds 

between 3’-C or 3’-U 

residues and the 

5’-OH residue of an 

adjacent nucleotide. 

Cleavage of 


between 3’-G residues 

and the 5’-OH residue of 

an adjacent nucleotide. 

Combines the activities of 

RNase A and RNase T1. 

Cleavage of all 

phosphodiester bonds in 

an RNA template. 

Cleavage of RNA 

phosphodiester bonds in 

RNA-DNA hybrids. 

Cleavage of all 


in RNA. 

Substrate 

ssRNA 

ssRNA 

ssRNA 

Reaction 

products 

3’-CMP, 

3’-UMP, 


with terminal 

3’-CMP, 3’-UMP 

3’-GMP, 


with terminal 

3’-GMP 

3’-CMP, 

3’-UMP, 

3’-GMP, 


with terminal 

3’-CMP, 3’-UMP 

or 3’-GMP 

ssRNA 3’-NMP 

RNA in 

RNA-DNA 

hybrids 

dsRNA 

5’-P 


25-27 bp 

fragments 


Spin column 

or phenol/ 

chloroform 

extraction 

Spin column 

or phenol/ 

chloroform 

extraction 

Spin column 

or phenol/ 

chloroform 

extraction 

Heating at 

100°C, 30 min 

Heating at 

65°C, 10 min 

Spin column 

or phenol/ 

chloroform 

extraction 

EN0531 262 

EN0541/2 263 

EN0551 264 

EN0601/2 265 

EN0201/2 266 

F-602S/L 267 


262 

RNase A, DNase and Protease-free 

#EN0531 10 mg (10 mg/ml) 


RNase T1 p.263 

RNase A/T1 Mix p.264 

RNase I p.265 

Proteinase K (recombinant), PCR grade p.270 

Plasmid Miniprep Kit p.304 

Genomic DNA Purification Kit p.311 

 

 

 

 



Description 

RNase A is an endoribonuclease that specifically 

degrades single-stranded RNA at C and U 

residues. 

The enzyme cleaves the phosphodiester bond 

between the 5’-ribose group of a particular 

nucelotide and the phosphate group attached 

to the 3’-ribose of an adjacent pyrimidine 

nucleotide. The resulting 2’, 3’-cyclic 

phosphate is hydrolyzed to the corresponding 

3’-nucleoside phosphate (1, 2). 

Features 

Guaranteed DNase free not necessary to heat 

RNase A for optimal activity. 

Applications 

Plasmid and genomic DNA preparation (3, 4). 

Removal of RNA from recombinant protein 

preparations. 

Ribonuclease protection assays. Used in 

conjunction with RNase T1 (3). 

Mapping single-base mutations in DNA or 

RNA (5, 6). 

Concentration 

10 mg/ml. 

Protein concentration is determined 

by measuring the absorbance at 278 

nm using molar absorption coefficient 

=9800 M -1 cm -1 (7). 

Source 

Bovine pancreas. 



5’ pPupPupPupPypPypPupPupPupPupPypPupPypPupPupPu 3’ 

ssRNA 

pPupPupPupPyp + Pyp + PupPupPupPupPyp + PupPyp + PupPupPu 

Pu – purine ribonucleoside (i.e. A, G) 

Py – pyridine ribonucleoside (i.e. C, U) 

p – phosphate 

RNase A activity. 


One unit of the enzyme causes an increase in 

absorbance of 1.0 at 260 nm when yeast RNA 

is hydrolyzed at 37°C and pH 5.0. 

Fifty units are approximately equivalent to 

1 Kunitz unit (8). 

Specific Activity 

>5000 u/mg protein (>100 Kunitz units/mg 

protein). 



50 mM Tris-HCl (pH 7.4) and 50% (v/v) glycerol. 


 

mamalian ribonuclease inhibitor, e.g., 

RiboLock RNase Inhibitor (#EO0381). 

Other inhibitors: 

uridine 2’,3’-cyclic vanadate, 

5’-diphosphoadenosine 3’-phosphate and 

5’-diphosphoadenosine 2’-phosphate (2), 

SDS, diethyl pyrocarbonate, 

4 M guanidinium thiocyanate plus 0.1 M 

2-mercaptoethanol and heavy metal ions. 

 

spin column or phenol/chloroform extraction. 

Note 

 

1-100 μg/ml depending on the application. 

 

range of reaction conditions. At low salt 

concentrations (0 to 100 mM NaCl), RNase 

A cleaves single-stranded and doublestranded 

RNA as well the RNA strand 

in RNA-DNA hybrids. However, at NaCl 

concentrations of 0.3 M or higher, RNase A 

specifically cleaves single-stranded RNA (9). 

References 

1. Blackburn, P., Moore S., Pancreatic ribonuclease, 

The Enzymes, V, (Boyer, P.D, Ed.), Academic Press, New 

York, the third edition, vol. 15, 317-433, 1982. 

2. Raines, R.T., Ribonuclease A, Chem. Rev., 98, 

1045-1065, 1998. 

3. 

Laboratory Manual, the third edition, Cold Spring Harbor 

Laboratory Press, Cold Spring Harbor, New York, 1.31- 

1.38, 2001. 

4. Sharma, R.C., et al., A rapid procedure for isolation 

of RNA-free genomic DNA from mammalian cells, 


5. Myers R.M., et al., Detection of single base substitutions 

by ribonuclease cleavage at mismatches in RNA:DNA 

duplexes, Science 230, 1242-1246, 1985. 

6. Winter E., et al., A method to detect and characterize 

point mutations in transcribed genes: Amplification and 

overexpression of the mutant c-Ki-ras allele in human 

tumor cells, Proc. Natl. Acad. Sci. USA, 82, 7575-7579, 

1985. 

7. Sela, M., Anfinsen, C.B., Some spectrophotometric and 

polarimetric experiments with ribonuclease, Biochim. 

Biophys. Acta, 24, 229-235, 1957. 

8. Kunitz, M.A., A spectrophotometric method for the 

 

563-568, 1946. 


 

York, 3.13.1, 1994-2005.

RNase T1 

#EN0541 100,000 u (1000 u/μl) 

#EN0542 500,000 u (1000 u/μl) 


RNase A p.262 




 

 

 

 



References 

1. Takahashi K., Moore S., Ribonuclease T1, The Enzymes, 

V, (Boyer, P.D, Ed.), Academic Press, New York, the third 

edition, vol. 15, 435-468, 1982. 

2. 



3. Sawadogo, M., Roeder, R.G., Factors involved in specific 

transcription by human RNA polymerase II: Analysis by a 

rapid and quantitative in vitro assay, Proc. Natl. Acad. Sci. 

USA, 82, 4394-4398, 1985. 



Description 

RNase T1 is an endoribonuclease that 

specifically degrades single-stranded RNA at 

G residues. It cleaves the phosphodiester 

bond between 3’-guanylic residue and the 

5’-OH residue of adjacent nucleotide with the 

formation of corresponding intermediate 

2’, 3’-cyclic phosphate (1). The reaction 

products are 3’-GMP and oligonucleotides with 

a terminal 3’-GMP. 

RNase T1 does not require metal ions for activity. 

Applications 

Removal of RNA from DNA preparations. 

RNA sequencing (1). 

Ribonuclease protection assays. Used in 

conjunction with RNase A (2). 


preparations. 

Determination of the level of RNA transcripts 

synthesized in vitro from DNA templates 

containing a “G-less cassette” (3). 

Concentration 

1000 u/μl 

5’ pNpNpNpGpGpNpNpNpNpGpNpGpNpNpN 3’ 

ssRNA 

pNpNpNpGp + GMP + NpNpNpNpGp 

+ NpGp + NpNpN 

N – any ribonucleoside, except G (i.e. A, C or U) 

G – guanosine 


RNase T1 activity. 

Source 

E.coli cells with a cloned rntA gene of 

Aspergillus oryzae. 





absorbance of 1.0 at 260 nm in 15 min when 

yeast RNA is hydrolyzed at 37°C and pH 7.5. 


mixture: 50 mM Tris-HCl (pH 7.5), 2 mM EDTA, 

3 mg/ml yeast RNA. 



50 mM Tris-HCl (pH 7.4) and 50% (v/v) glycerol. 


2+ , Ca2+ , Zn2+ , Fe2+ , 

Cu2+ (MgCl2 at 100 mM concentration is 

approx. 40% inhibitory, CaCl2 at 10 mM is 

approx. 30% inhibitory); 

mononucleotides (2’-GMP, 3’-GMP, etc.); 

guanilyl-2’,5’-guanosine is a specific 

inhibitor (4). 

 

reliably removed by spin column or phenol/ 

chloroform extraction. 

Recommendations for Use 

Reaction mixture for complete digestion of RNA: 

50 mM Tris-HCl (pH 7.5), 1 mM EDTA and 0.2 u RNase T1/μg RNA. Incubate at 37°C for 30 min. 

RNase digestion mixture for RNase protection assay (2): 

10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5 mM EDTA (pH 7.5), 40 μg/ml RNase A, 1000 u/ml RNase T1. 


264 

RNase A/T1 Mix 

#EN0551 1 ml 

(2 mg/ml of RNase A and 

5000 u/ml of RNase T1) 






 

 

 

 


Description 

RNase A/T1 Mix combines the RNA degradation 

activity of both RNase A and RNase T1. The 

RNase A specifically hydrolyzes RNA at C and U 

residues; RNase T1 specifically hydrolyzes RNA 

at G residues (1). 

Features 

Higher level RNA degradation than with 

RNase A and RNase T1 separately. 

Guaranteed DNase free not necessary to heat 

the mix for optimal activity. 

Applications 

Removal of RNA from DNA preparations. 


preparations. 

Ribonuclease protection assays 

(see recommendations for use bellow). 

Concentration 

2 mg/ml (approx. 10,000 u/ml (200 Kunitz u/ml)) 

of RNase A, 5000 u/ml of RNase T1. 

5’ pBpBpBpApApBpBpBpBpApBpApBpBpB 3’ 

ssRNA 

5’ pBp + 3’ BMP + ApApBp+ ApBp + B 

B – pyridine ribonucleoside (i.e. C, U) or 

guanoside (i.e. G) 

A – adenosine 


RNase A/T1 Mix activity. 


RNase digestion mixture for RNase protection assay: 

10 mM Tris-HCl (pH 7.5), 300 mM NaCl, 

5 mM EDTA (pH 7.5), 20 μl of RNase A/T1 Mix per 1 ml of reaction mixture. 

Source 

RNase A: Bovine pancreas. 

RNase T1: E.coli cells with a cloned rntA gene 

of Aspergillus oryzae. 

Unit Definition for RNase A 


absorbance of 1.0 at 260 nm when yeast RNA 

is hydrolyzed at 37°C and pH 5.0. 

Fifty units are approximately equivalent to 

1 Kunitz unit (3). 

Unit Definition for RNase T1 


absorbance of 1.0 at 260 nm in 15 min when 

yeast RNA is hydrolyzed at 37°C and pH 7.5. 


The enzymes are supplied in: 50 mM Tris-HCl 

(pH 7.4) and 50% (v/v) glycerol. 


see pp.262-263. 

 

by spin column or phenol/chloroform extraction. 

References 


 

York, 3.13.1-3.13.3, 1994-2005. 

2. 



3. Kunitz, M.A., A spectrophotometric method for the 

 

563-568, 1946.

RNase I 

#EN0601 1000 u (10 u/μl) 

#EN0602 5000 u (10 u/μl) 





Bovine Serum Albumin (BSA) p.422 


 

 

 

 

References 

1. Shen, V., Schlessinger, D., RNase I of Escherichia coli, The 

Enzymes (Boyer, P.D., Ed.), Academic Press Inc., New York, 

vol. 15B, 503-506, 1982. 

2. Zhu, L., Deutscher, M.P., The Escherichia coli rna gene 

encoding RNase I: sequence and unusual promoter 

structure, Gene, 119, 101-106, 1992. 

3. 



Description 

Ribonuclease I (RNase I), an endoribonuclease, 

preferentially hydrolyzes single-stranded RNA to 

nucleoside 3’-monophosphates via nucleoside 

2’,3’-cyclic monophosphate intermediates. The 

enzyme does not require any metal ions for 

activity. 

Features 

Stable and active under a wide variety of 

reaction conditions. 

Can be heat inactivated in 30 min at 100°C. 

Applications 

Removal of RNA from DNA solutions (2). 


preparations. 

Ribonuclease protection assays (3). 

Concentration 

10 u/μl 

Source 

E.coli cells with a cloned rna gene of E.coli. 



5’ 3’ 

ssRNA 

RNase I activity. 

5’ NMPs 


Removal of RNA from DNA solutions. 

1. Add 15-20 u of RNase I per 1 μg of RNA. RNase I is 90% active within pH 

range 7.0-8.8 at salt concentration 100-200 mM. Incubation with RNase I 

can be performed simultaneously with the digestion of DNA by restriction 

endonucleases. 

2. Incubate at 37°C for 30 minutes. 

3. Purify DNA by spin column or phenol/chloroform extraction. 


One unit of the enzyme catalyzes degradation of 

100 ng of E.coli ribosomal RNA per second into 

acid-soluble nucleotides at 37°C. 


mixture: 20 mM Tris-acetate (pH 8.0), 

100 mM NaCl, 0.1 mM EDTA, 

0.01% (v/v) Triton X-100, 

40 μM E.coli ribosomal [ 3 H]-RNA. 






 

irreversibly inactivates the enzyme. 

 



Note 

 

effect on RNase I. 

 

 

the RNase T2 family. 

 

not inhibit RNase I, and may even slightly 

stimulate its activity and stabilize it against heat 

inactivation. Triton X-100 or BSA (at 0.1 mg/ 

ml) may prevent RNase I from sticking to glass 

vessels when working with dilute solutions. 

 


266 

RNase H 

#EN0201 100 u (5 u/μl) 

#EN0202 500 u (5 u/μl) 







0.5 M EDTA, pH 8.0 p.421 



Description 

Ribonuclease H (RNase H) specifically degrades 

the RNA strand in RNA-DNA hybrids. It does 

not hydrolyze the phosphodiester bonds within 

single-stranded and double-stranded DNA and 

RNA. 

Applications 

Removal of mRNA prior to synthesis of second 

strand cDNA (1). 

RT-PCR and RT-qPCR: removal of RNA after 

first strand cDNA synthesis. 

Removal of the poly(A) sequences of mRNA 

after hybridization with oligo(dT) (2). 

Site-specific cleavage of RNA (3). 

Studies of in vitro polyadenylation reaction 

products (4). 

Concentration 

5 u/μl 

Source 

E.coli cells with a cloned rnh gene of E.coli. 

RNA 5’ RpRpRpRpRpRpRpRpRp 3’ 

DNA 3’ DpDpDpDpDpDpDpDpDp 5’ 

RNA/DNA duplex 

RpR + pRpRpRpR + pRpRpRp+ ssDNA 

R – ribonucleoside 

D – deoxyribonucleoside 


RNase H activity. 




One unit of the enzyme catalyzes the formation of 

1 nmol of acid soluble products in 20 min at 37°C. 


mixture: 20 mM Tris-HCl (pH 7.8), 40 mM KCl, 

8 mM MgCl2, 1 mM DTT, 

24 μM [ 3H]-poly(A)·poly(dT), 0.03 mg/ml BSA, 




25 mM HEPES-KOH (pH 8.0), 50 mM KCl, 







 

reagents. 

 

References 

1. 

method for generating cDNA libraries, Gene, 25, 

263-269, 1983. 

2. Davis, R., et al., Tandemly repeated exons encode 

81-base repeats in multiple, developmentally regulated 

Schistosoma mansoni transcripts, Mol. Cell Biol., 8, 

4745-4755, 1988. 

3. Donis-Keller, H., Site specific enzymatic cleavage of RNA, 


4. Goodwin, E.C., Rottman, F.M., The use of RNase H and 

poly(A) junction oligonucleotides in the analysis of in vitro 

polyadenylation reaction products, Nucleic Acids Res., 20, 

916, 1992.


(Not available in USA) 

#F-602S 60 u (1 u/μl) 


5x PowerCut Dicer Reaction Buffer 500 μl 

#F-602L 300 u (1 u/μl) 


5x PowerCut Dicer Reaction Buffer 500 μl 


Replicator RNAi Kit p.329 

Phi6 dsRNA p.387 

NTP Set p.411 

dNTPs p.408 

GeneRuler Ultra Low Range DNA Laders p.359 

T7 RNA Polymerase p.330 


Phi6 RNA replicase p.332 

References 

1. Calegari, F. et al., Tissue-specific RNA interference in 

postimplantation mouse embryos with endoribonucleaseprepared 

short interfering RNA, Proc. Natl. Acad. Sci. USA, 

99, 14236-14240, 2002. 

 

large dsRNAs into siRNAs suitable for gene silencing, Nat. 

Biotechnol., 21, 324-328, 2003. 

3. Yang D. et al., Short RNA duplexes produced by hydrolysis 

with Escherichia coli RNase III mediate effective RNA 

interference in mammalian cells, Proc. Natl. Acad. Sci. 

USA, 99, 9942-9947, 2002. 

Description 

PowerCut Dicer is a novel recombinant 

endoribonuclease originating from Giardia 

intestinalis. It cleaves dsRNA to small interfering 

RNA (siRNA) with a 100% efficiency and 

outperforms all other available siRNA producing 

enzymes. The resulting siRNA fragments are 

25-27 nucleotides long and are capable of 

triggering RNA interference when transfected 

into cells. Therefore the siRNA fragments 

generated with the PowerCut Dicer enzyme are 

ideal for use in gene silencing studies. Pooled 

siRNAs the siRNA fragments generated with the 

PowerCut Dicer enzyme are also preferred over 

synthetic siRNA oligos if the target sequence 

cannot be specified well enough or when the 

sequence is prone to variations (e.g., viral 

targets). 

PowerCut Dicer combined with the Replicator 

RNAi Kit are a powerful combination for 

generating high amounts of siRNA from 

in vitro-produced dsRNA. 

Features 

100% cleavage efficiency for maximal siRNA 

yields. 

Will not overdigest siRNAs. 

Produces 25-27 nucleotide-long siRNAs with 

strong silencing function. 

Free of contaminating RNase, endo- and 

exonuclease activities. 



Application 

For production of siRNA ideal for gene silencing 

studies. 

Source 

E.coli cells with a cloned gene of PowerCut 

Dicer gene from Giardia intestinalis. 

Concentration 

1 u/μl 


One unit is defined as the amount of enzyme that 

is needed to completely cleave 1 μg of 192 bp 

double stranded RNA substrate to siRNA 

in 16 hours at 37°C. 


Enzyme is supplied in: 50 mM Tris-HCl 

(pH 8.0 at 5°C), 50 mM NaCl, 5 mM MgCl2, 

0.2 mg/ml BSA, 50% glycerol. 

Conventional 

dicer 

RNase III 

M 1 2 3 1 2 3 1 2 3 

Demonstration of efficient generation of siRNAs without 

overdigestion using the Thermo Scientific PowerCut Dicer. 

Three different dsRNA substrates (Furin, eGFP and Phi6 dsRNA) were 

cleaved using PowerCut Dicer and two other commercial enzymes used 

for producing siRNAs (from major suppliers). Reactions were performed 

according to the manufacturers’ instructions. PowerCut Dicer cleaved 

all the three substrates with 100% efficiency. The conventional dicer 

gave only partial digestion, whereas RNase III also digested part of the 

produced siRNAs resulting in lower yield. 

1 – Furin dsRNA cleaved with appropriate enzyme. 

2 – eGFP dsRNA cleaved with appropriate enzyme. 

3 – Phi6 dsRNA cleaved with appropriate enzyme. 

M – a 27-bp RNA marker. 


268 

Sugar Nonspecific Nucleases 

Table 2.6. Properties of sugar nonspecific nucleases. 

Nuclease Applications Reaction catalyzed Substrate Reaction products Inactivation Cat. # Page 

Micrococcal 

Nuclease 

S1 Nuclease 


 

crude cell-free extracts. 

 

 

 

for structure-function studies. 

 

 

 

 

deletions in DNA fragments. 

* – the enzyme activity is strictly dependent on Ca 2+ . 



dNMP – deoxyribonucleoside monophosphate. 


Micrococcal Nuclease 

Degradation of nucleic 

acids preferentially at AT or 

AU-rich regions. 

Degradation of ss nucleic 

acids. Five times more 

active on DNA than on RNA. 

DNA 

RNA 

ssDNA 

ssRNA 

#EN0181 8000 u (300 u/μl) Description 

Micrococcal Nuclease (S7 Nuclease) is a 

relatively nonspecific endo-exonuclease that 

digests single-stranded and double-stranded 

nucleic acids, but is more active on singlestranded 

substrates. Cleavage of DNA or RNA 

occurs preferentially at AT or AU-rich regions 

yielding mononucleotides and oligonucleotides 

with terminal 3’-phosphates. The enzyme 

activity is strictly dependent on Ca 2+ . 

5’ 

3’ 

dsDNA 

5’ 

ssDNA/RNA 

Ca 2+ 

3’dNMPs + 3’oligonucleotides 

Ca 2+ 

Micrococcal nuclease activity. 

3’ 

5’ 

3’NMPs or 3’dNMPs + 3’oligonucleotides 

3’ 

Applications 

Hydrolysis of nucleic acids in crude cell-free 

extracts (1). 

Sequencing of RNA (2). 

Studies of chromatin structure (3). 

Protein folding and structure-function 

studies (4, 5). 

Concentration 

300 u/μl 

Source 

E.coli cells with a cloned nuc gene encoding 

Staphylococcus aureus extracellular nuclease 

(micrococcal nuclease). 

3’-dNMPs 


3’-NMPs 


5’-dNMPs 


5’-NMPs 


Addition of 

EGTA or EDTA* 

Heating at 

70°C for 

10 min in the 

presence of 

EDTA 

EN0181 268 

EN0321 269 




One unit of the enzyme releases 1.0 A260 unit of 

acid-soluble products in 30 min at 37°C. 


mixture: 100 mM sodium-glycine (pH 8.6), 

10 mM CaCl2 and 4.5 mM calf thymus DNA. 



20 mM Tris-HCl (pH 7.6), 50 mM NaCl and 



 

3’ 5’-diphosphate. 

 

References 

1. 

dependent translation system from reticulocyte lysates, 

 

2. 

from Staphylococcus aureus and Neurospora crassa 

discriminate between uridine and cytidine, Nucleic Acids 

Res., 6, 3481-3490, 1979. 

3. 

 

21, 127-137, 1989. 

4. Tucker, P.W., et al., Staphylococcal nuclease reviewed: 

a prototypic study in contemporary enzymology. IV. The 

nuclease as a model for protein folding, Molec. Cell. 

Biochem., 23, 131-140, 1979. 

5. Torchia, D.A., et al, Staphylococcal nuclease: sequential 

assignments and solution structure, Biochemistry, 27, 

5509-5524, 1989.

S1 Nuclease 

#EN0321 10,000 u (100 u/μl) 




Exonuclease III (Exo III) p.259 


References 

1. Lehman, R.I., Endonucleases specific for single-stranded 

polynucleotides, The Enzymes, 3rd. Ed. (Boyer, P.D., Ed.), 

4,193-201,1981. 

2. Roberts T.M. et al., A general method for maximizing the 

expression of a cloned gene, Proc. Natl. Acad. Sci. USA, 

76, 760-764, 1979. 

3. 

simian virus 40, Proc. Natl. Acad. Sci. USA, 75, 

1274-1278, 1978. 

4. Weidle, U., Weissmann, C., The 5’-flanking region of 

a human IFN-alpha gene mediates viral induction of 

transcription, Nature, 303, 442-446, 1983. 

5. Henikoff, S., Unidirectional digestion with exonuclease III 

creates targeted breakpoints for DNA sequencing, Gene, 

28, 351-359, 1984. 

6. Vogt, V.M., Purification and further properties of singlestrand-specific 

nuclease from Aspergillus oryzae, 

 

Description 

S1 Nuclease degrades single-stranded nucleic 

acids, releasing 5’-phosphoryl mono- or 

oligonucleotides. It is five times more active on 

DNA than on RNA (1). S1 Nuclease also cleaves 

dsDNA at the single-stranded region caused 

by a nick, gap, mismatch or loop. S1 Nuclease 

exhibits 3’-phosphomonoesterase activity. 

This glycoprotein enzyme contains a 

carbohydrate content of 18%. 

Applications 

Removal of single-stranded overhangs from 

DNA fragments (2). 

S1 transcript mapping (3, 4). 

Cleavage of hairpin loops. 

Creation of unidirectional deletions in DNA 

fragments in conjunction with Exo III (5). 

Concentration 

100 u/μl 

Source 

Aspergillus oryzae. 

5’ 

5’ 

3’ 

3’ 

dsDNA 

5’ 

3’ 

5’ 

ssDNA/RNA 

5’ 

3’ 

dsDNA/RNA 

Nuclease S1 activities. 

Zn 2+ 

Zn 2+ 

5’dNMPs or rNMPs 

High enzyme concentration 

Zn 2+ 

5’dNMPs or rNMPs 

Moderate enzyme concentration 

5’ 

3’ 

nicked DNA 

3’ 

5’ 

+ 

Zn 2+ 

5’ 

3’ 

3’ 

3’ 

5’ 

3’ 

3’ 

5’ 

3’ 

5’ 

5’ 

3’ 

5’ 




One unit of the enzyme produces 1 μg of acid 

soluble deoxyribonucleotides in 1 min at 37°C. 


mixture: 30 mM sodium acetate (pH 4.5), 

50 mM NaCl, 0.1 mM ZnCl2, 5% (v/v) glycerol, 

800 μg/ml heat denatured calf thymus DNA. 




0.1 mM ZnCl2 and 50% (v/v) glycerol. 


200 mM sodium acetate (pH 4.5 at 25°C), 

1.5 M NaCl and 10 mM ZnSO4. Inhibition and Inactivation 

i, Pi, 5’-ribonucleotides and deoxyribonucleotides 

 

the presence of EDTA. 

Note 

 

double-stranded DNA, RNA and DNA/ 

RNA hybrids at high enzyme and low salt 

concentrations (6). 


» 3.2.3. Removal of 3’- and 5’-overhangs 

with S1 Nuclease p.295 


270 

Other Products 

Proteinase K (recombinant), PCR grade 

#EO0491 1 ml 

(>600 u/ml, ~20 mg/ml) 

#EO0492 5x1 ml 

(>600 u/ml, ~20 mg/ml) 



Water, nuclease-free 

 

p.420 

DNA Purification Kit p.312 

 

 


» 4.1. Purification of genomic DNA from 

mouse tail with Proteinase K p.322 

» 4.2. Purification of DNA from cultured 

eukaryotic cells with Proteinase K p.322 


Description 

Proteinase K is an endolytic protease that 

cleaves peptide bonds at the carboxylic sides of 

aliphatic, aromatic or hydrophobic amino acids. 

Proteinase K is classified as a serine protease 

(1). The smallest peptide to be hydrolyzed by 

this enzyme is a tetrapeptide. 

Features 

Ready-to-use solution. 

Active in a wide range of reaction conditions. 

Applications 

Isolation of genomic DNA from mouse tail. 

Isolation of genomic DNA from cultured cells. 

Removal of DNases and RNases when isolating 

DNA and RNA from tissues or cell lines (2, 3). 

Determination of enzyme localization (4). 

Improving cloning efficiency of PCR 

products (5). 

Concentration 

Approx. 600-1000 u/ml 

14-22 mg/ml 

Source 

Pichia pastoris cells with a cloned gene from 

Tritirachium album. 


28.9 kDa monomer (6). 


One unit of the enzyme liberates Folin-positive 

amino acids and peptides corresponding to 

1 μmol tyrosine in 1 min at 37°C using 

denatured hemoglobin as substrate. 


mixture: 0.08 M potassium phosphate (pH 7.5), 

5 M urea, 4 mM NaCl, 3 mM CaCl 2 and 

16.7 mg/ml hemoglobin. 

20’ 



10 mM Tris-HCl (pH 7.5), 

containing calcium acetate and 



Inhibitors: Proteinase K is not inactivated by 

metal chelators, by thiol-reactive reagents or 

by specific trypsin and chymotrypsin inhibitors. 

Phenylmethylsulfonyl fluoride and diisopropyl 

phosphorofluoridate completely inhibit the 

enzyme (1). 

 

Note 

The recommended working concentration of 

Proteinase K is 0.05-1 mg/ml. The activity of 

the enzyme is stimulated by 0.2-1% SDS or 

by 1-4 M urea (3). 

2+ protects Proteinase K against autolysis, 

increases the thermal stability and has a 

regulatory function for the substrate binding 

site of Proteinase K (7). 

 

optimum pH 7.5-8.0 (8). 

References 

1. Ebeling, W., et al., Proteinase K from Tritirachium album 

 

2. Wiegers, U., Hilz, H., A new method using ‘proteinase 

K’ to prevent mRNA degradation during isolation from 

HeLa cells, Biochem. and Biophys. Res. Commun., 44, 

513-519, 1971. 

3. Hilz, H., et al., Stimulation of proteinase K action by 

denaturing agents: application to the isolation of nucleic 

 

Biochem., 56, 103-108, 1975. 

4. Brdiczka, D., Krebs, W., Localization of enzymes by means 

of proteases, Biochim. Biophys. Acta, 297, 203-212, 1973. 

5. 

polymerase chain reaction (PCR) products after proteinase 

K digestion, Nucleic Acids Res., 19, 184, 1991. 

6. 

from mold Tritirachtum album Limber Proteinase K – a 

subtilisin related enzyme with disulfide bonds, FEBS Lett., 

199, 139-144, 1986. 

7. 

 

8. 

domain inhibits eight different serine proteinases of varied 

specificity on the same... Leu18-Glu19... reactive site, 

Biochemistry, 24, 5313-5320, 1985.

RiboLock RNase Inhibitor 

#EO0381 2500 u (40 u/μl) 

#EO0382 4x2500 u (40 u/μl) 

#EO0384 24x2500 u (40 u/μl) 





NTPs p.411 

DTT p.420 

DEPC-treated Water p.420 

M C 1 C 2 1 2 3 4 

Inhibition of RNase A by Thermo Scientific 

RiboLock RNase Inhibitor. 

Total human RNA (1 μg) was incubated at 37°C with 

20 u of RiboLock RNase Inhibitor and increasing 

amounts of RNase A (#EN0531). 

M – RiboRuler High Range RNA Ladder (#SM1821). 

C 1 – total human RNA. 

C 2 – total human RNA with RNase A (no RiboLock added). 

1-4 – total human RNA with RiboLock and RNase A. 

References 

1. Nielsen, D.A., Shapiro 

transcripts using T7 RNA polymerase, Nucleic Acids Res., 

14, 5936, 1986. 

2. Martynoff, G., et al., Synthesis of a full length DNA 

complementary to thyroglobulin 33S messenger RNA, 

Biochem. Biophys. Res. Commun., 93, 645-653, 1980. 

3. Scheele, G., Blackburn, P., Role of mammalian RNase 

inhibitor in cell-free protein synthesis, Proc. Natl. Acad. 

Sci. USA, 76, 1898-1902, 1979. 

4. Van Gelder., et al., Amplified RNA synthesized from limited 

quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. 

USA, 87, 1663-1667, 1990. 

5. Eichler, D.C., et al., Effect of human placental ribonuclease 

inhibitor in cell-free ribosomal RNA synthesis, Biochem. 

Biophys. Res. Commun., 101, 396-403, 1981. 

6. 

anti-angiogenic properties and leads to reduced tumor 

 

Description 

RiboLock RNase Inhibitor inhibits the activity 

of RNases A. B and C by binding them in a 

noncompetitive mode at a 1:1 ratio. RiboLock 

does not inhibit eukaryotic RNases T1, T2, U1, 

U2, CL3 or prokaryotic RNases I and H. 

Features 

Protects RNA from degradation at 

temperatures up to 55°C. 

Performs under a wide range of reaction 

conditions. 

Applications 

Inhibition of RNA degradation in the following: 

– in vitro transcription (1), 

– cDNA synthesis (2), 

– in vitro translation (3), 

– isolation of mammalian cell fractions that 

contain mRNA-protein complex (3), 

– RNA amplification (4). 

RNA purification and storage. 

Separation and identification of specific 

ribonuclease activities (5). 

Studies of tumor suppression (6). 

M C1 C2 1 2 3 4 5 

Thermostability of Thermo Scientific RiboLock 

RNase Inhibitor. 

Total human RNA (1 μg) was incubated with 20 u of 

RiboLock RNase Inhibitor and 50 pg of RNase A 

(#EN0531), and incubated at increasing temperatures. 

M – RiboRuler High Range RNA Ladder (#SM1821). 

C1 – total human RNA. 

C2 – total human RNA with RNase A 

(no RiboLock added). 

1-5 – total human RNA with RiboLock and RNase A. 

Concentration 

40 u/μl 

Source 

E.coli cells with a cloned gene encoding 

mammalian ribonuclease inhibitor. 




One unit of the RiboLock RNase Inhibitor inhibits 

the activity of 5 ng RNase A by 50%. 

Inhibitor activity is assayed in the following 


1.2 mM EDTA, 0.1 mg/ml BSA, 

100 ng/ml RNase A, 0.1 mg/ml [ 3H]-RNA, 10 mg/ml yeast RNA, 8 mM DTT. 


The protein is supplied in: 

20 mM HEPES-NaOH (pH 7.5), 

50 mM NaCl, 8 mM DTT, 

0.015% (v/v) ELUGENT Detergent and 



Inhibitors: common denaturants 

(SDS, urea and all oxidizing reagents 

(p-chloromercuribenzoate, dissolved oxygen, 

ions in their higher oxidation states) strongly 

inhibit 

RiboLock RNase Inhibitor and release the 

RNase bound. 

 

Residual activity detectable after 10 min 

heating at 70°C. 

Note 

 

stability during long term storage, but is not 

necessary for inhibitor activity. 

 

reaction mixture. 


» 5.3. In vitro transcription p.335 

» 5.4. Synthesis of radiolabeled RNA probes 

of high specific activity p.335 

» 6.4.1. Synthesis of radiolabeled RNA probes 


» 6.5. Synthesis of labeled cDNA p.350 


272 

Pyrophosphatase, Inorganic (from yeast) 

#EF0221 10 u (0.1 u/μl) 


Storage (Dilution) Buffer 1 ml 



phi29 DNA Polymerase p.245 


Klenow Fragment, exo – p.248 



Terminal Deoxynucleotidyl Transferase (TdT) p.251 

Terminal Transferase p.252 



Description 

Inorganic Pyrophosphatase catalyzes the 

hydrolysis of inorganic pyrophosphate to two 

orthophosphates. The enzyme requires a 

divalent metal cation, with Mg 2+ conferring the 

highest activity (1). 

Features 

Active in Thermo Scientific buffers for DNA 

Polymerases, RNA Polymerases and Reverse 

Transcriptases. 

Applications 

High yield RNA synthesis by in vitro transcription (2). 

DNA polymerization reactions: preventing 

accumulation of pyrophosphate (3, 4). 

Removal of contaminant PPi in reagents used 

for SNP genotyping by methods based on the 

detection of pyrophosphate (5). 

Concentration 

0.1 u/μl 

Source 

E.coli cells with a cloned ppa gene of 

Sacharomyces cerevisiae. 

polymerase 

Template + (d)NTPs 


This enzyme is homodimer. It consists of two 

identical subunits of 32 kDa. 


One unit of the enzyme hydrolyzes 1 μmol of 

inorganic pyrophosphate in 1 min at 25°C. 


mixture: 100 mM Tris-HCl (pH 7.2), 2 mM MgCl2 and 2 mM inorganic pyrophosphate (PPi). 

Storage (Dilution) Buffer 


10 mM Tris-HCl (pH 7.5), 0.1 mM EDTA and 



Inhibitors: imidodiphosphate, -glycol 

disphosphates, methanedial diphosphate, 

1,2-ethanedial diphosphate (6). 

 



polymerase 

pyrophosphatase 

3- 

Template + (d)NTPs Product + Template + (d)NTPs + 2 PO4 Use of Pyrophosphatase, in preventing inhibition of nucleic acid synthesis by 

phyrophosphate. 

Pyrophosphatase prevents inhibition of the polymerization reaction by hydrolysis of 

pyrophosphate accumulated during reaction. 



Note 

 

storage (dilution) buffer. 

Product + Template + (d)NTPs + PP i 

References 

1. Cooperman, B.S., The mechanism of action of yeast 

inorganic pyrophosphatase, Meth. Enzymol., 87, 

526-548, 1982. 

2. 

pyrophosphatase to improve the yield of in vitro 

transcription reactions catalyzed by T7 RNA polymerase, 

Biotechniques, 9, 713-714, 1990. 

3. Tabor, S., Richardson, C.C., DNA sequence analysis with 

a modified bacteriophage T7 DNA polymerase. Effect of 

 

8322-8328, 1990. 

4. Dean, B.F., et al., Rapid amplification of plasmid and 

phage DNA using phi29 DNA polymerase and multiplyprimed 

Rolling Circle amplification, Genome Res., 11, 

1095-1099, 2001. 

5. Zhou, G.H., et al., Quantitative detection of single 

nucleotide polymorphisms for a pooled sample by a 

bioluminometric assay coupled with modified primer 

extension reactions (BAMPER), Nucleic Acids Res., 29, 

E93, 2001. 

6.

Uracil-DNA Glycosylase (UDG, UNG) 

#EN0361 200 u (1 u/μl) 



#EN0362 5x200 u (1 u/μl) 




Exonuclease III (Exo III) p.259 

Endonuclease IV, E.coli (Endo IV) p.256 

dNTP/dUTP Mix p.408 

dUTP p.409 


References 

1. 

252, 10, 3286-3294, 1977. 

2. Longo, M.C., et al., Use of uracil DNA glycosylase to 

control carry-over contamination in polymerase chain 

reactions, Gene, 93, 125-128, 1990. 

3. Vaughan, P., McCarthy, T.V., A novel process for mutation 

detection using uracil DNA glycosylase, Nucleic Acids 

Res., 26, 810-815, 1998. 

4. Kunkel, T.A., Rapid and efficient site-specific mutagenesis 

without phenotypic selection, Proc. Natl. Acad. Sci. USA, 

82, 488-492, 1985. 

5. Devchand, P.R., et al., Uracil-DNA glycosylase as a probe 

for protein-DNA interactions, Nucleic Acids Res, 21, 

3437-3443, 1993. 

6. Booth, P.M., et al., Assembly and cloning of coding 

sequences for neurotrophic factors directly from genomic 

DNA using polymerase chain reaction and uracil DNA 

glycosylase, Gene, 146, 303-308, 1994. 

7. Serrano-Heras, G., et al., Protein p56 from the Bacillus 

subtilis phage phi29 inhibits DNA-binding ability of uracil- 

DNA glycosylase, Nucleic Acids Res, 13, 1-9, 2007. 

Description 

Uracil-DNA Glycosylase catalyzes the hydrolysis 

of the N-glycosylic bond between uracil and 

sugar, leaving an apyrimidinic site in uracil 

containing single- or double-stranded DNA. The 

enzyme has no activity on RNA (1). 

Features 


restriction enzymes and thermophilic 

polymerases. 

Applications 

Control of carry-over contamination in PCR (2). 

Glycosylase mediated single nucleotide 

polymorphism detection (GMPD) (3). 

Site-directed mutagenesis (4). 

As a probe for protein-DNA interaction 

studies (5). 

SNP genotyping. 


Generation of single strand overhangs on 

PCR products and cDNA. 

Concentration 

1 u/μl 

Source 

E.coli K12 cells. 

5’ 

3’ 

uracil containing 

dsDNA 

5’ 

3’ 

dsDNA with 

abasic site 

U – uracil 


a – abasic site 

uracil 

Uracil-DNA Glycosylase activity. 

U 

P-a-P 

3’ 

5’ 

3’ 

5’ 



+ 




One unit of the enzyme catalyzes the release 

1 nanomole of uracil from uracil-containing DNA 

template in 60 min at 37°C. 




1 mM EDTA, 1 mM DTT, 0.05% (v/v) Tween 20 




10 mM EDTA, 100 mM NaCl. 


 

Bacillus subtilis phage PBS2, protein p56 

from the Bacillus subtilis phage phi29 (7). 

 

Enzyme activity is partially restored at 

temperatures lower than 55°C. Therefore, 

put PCR products on ice after PCR and load 

directly on a gel. 

Note 

 

DNA Glycosylase may be subsequently 

cleaved by heat, alkali-treatment or 

endonucleases that cleave specifically at 

abasic sites. 

 

divalent cations. 





274 


2.1. Activity of DNA/RNA modifying enzymes in Thermo Scientific buffers 

Thermo Scientific DNA/RNA modifying enzymes 

are supplied with optimized reaction buffers. 

However, it is often convenient to use these 

enzymes in other buffers for experiments that 

involve multiple enzymatic reactions. 

Activity of DNA/RNA modifying enzymes in Thermo Scientific buffers. 

DNA/RNA 

modifying enzyme 

* – buffers were supplemented with 0.5 mM ATP, required for T4 DNA Ligase activity. 

** – the activity of this enzyme was compared to activity in buffer A (for forward reaction). 

ND – not determined. 


Fast- 

Digest / 

Fast- 

Digest 

Green 

B G O R Tango 2X 

Tango 

Enzyme activity in 1X buffers, % 

BamHI Ecl136II, 

SacI 

EcoRI KpnI 

Taq 

with 

KCl 

Taq with 

(NH4)2SO4 

T4 DNA Ligase* 75-100 100 100 75-100 75-100 75-100 75-100 75-100 50 75-100 100 75 75 75 100 

FastAP 

Thermosensitive 


T4 Polynucleotide 

Kinase** (T4 PNK) 

The table below lists activities of DNA/RNA 

modifying enzymes in common reaction buffers, 

supplied with Thermo Scientific enzymes. 

100 100 100 100 100 100 100 100 100 100 100 100 50 100 ND 

100 75-100 100 100 75-100 100 100 100 50-75 100 75-100 100 0 100 100 

DNA Polymerase I 100 25-50 75-100 100 100 100 100 100 50-75 100 50-75 100 100 100 ND 

Klenow Fragment 100 25-50 20-50 100 100 100 100 100 50-75 100 50-75 100 100 100 100 

Klenow Fragment, exo – 100 25-50 25-50 100 100 100 100 100 50-75 100 75-100 100 100 100 ND 

T4 DNA Polymerase 100 75-100 75-100 100 100 100 100 100 100 100 100 50 100 100 100 

T7 DNA Polymerase 100 75-100 100 100 100 100 100 75-100 75-100 100 100 ND ND ND ND 

Exonuclease I (Exo I) ND ND ND ND ND ND ND ND ND ND ND 100 100 ND ND 

Exonuclease III (Exo III) 0-25 100 25-50 0-25 0-25 25-50 0-25 0-25 100 0-25 100 ND ND ND ND 

Lambda Exonuclease ND ND ND ND ND ND ND ND ND ND ND 50-75 50-75 ND ND 

phi29 DNA Polymerase 100 25-100 100 100 100 100 100 100 50-75 100 50-75 25-50 75-100 75-100 25-100 

Bsm DNA Polymerase 100 25-50 75-100 100 100 100 100 100 0-25 100 25-50 100 100 100 50-75 

RT 

T4 DNA 

Ligase

Buffer composition. 

Buffer 1X buffer composition 

B 10 mM Tris-HCl (pH 7.5 at 37°C) 10 mM MgCl 2 – – – 0.1 mg/ml BSA – – 

G 10 mM Tris-HCl (pH 7.5 at 37°C) 10 mM MgCl 2 50 mM NaCl – – 0.1 mg/ml BSA – – 

O 50 mM Tris-HCl (pH 7.5 at 37°C) 10 mM MgCl 2 100 mM NaCl – – 0.1 mg/ml BSA – – 

R 10 mM Tris-HCl (pH 8.5 at 37°C) 10 mM MgCl 2 100 mM KCl – – 0.1 mg/ml BSA – – 

Tango 33 mM Tris-acetate (pH 7.9 at 37°C) 10 mM Mg-acetate 66 mM K-acetate – – 0.1 mg/ml BSA – – 

2X Tango 66 mM Tris-acetate (pH 7.9 at 37°C) 20 mM Mg-acetate 132 mM K-acetate – – 0.2 mg/ml BSA – – 

BamHI 10 mM Tris-HCl (pH 8.0 at 37°C) 5 mM MgCl 2 100 mM KCl – 0.02% Triton X-100 0.1 mg/ml BSA – 1 mM BME 

Ecl136II, SacI 

10 mM Bis-Tris Propane-HCl 

(pH 6.5 at 37°C) 

2.2. Dilution of DNA/RNA modifying enzymes 

DNA/RNA modifying enzymes are supplied in 

their optimal storage buffers which are specially 

formulated for long term storage. 

10 mM MgCl 2 – – – 0.1 mg/ml BSA – – 

EcoRI 50 mM Tris-HCl (pH 7.5 at 37°C) 10 mM MgCl 2 100 mM NaCl – 0.02% Triton X-100 0.1 mg/ml BSA – – 

KpnI 10 mM Tris-HCl (pH 7.5 at 37°C) 10 mM MgCl 2 – – 0.02% Triton X-100 0.1 mg/ml BSA – – 

Taq with KCl 10 mM Tris-HCl (pH 8.8 at 25°C) 1.5 mM MgCl 2 50 mM KCl – 0.08% Nonidet P40 – – – 

Taq with 

(NH 4) 2SO 4 

75 mM Tris-HCl (pH 8.8 at 25°C) 2 mM MgCl 2 – – 0.01% Tween 20 – 20 mM (NH4) 2SO 4 – 

RT 50 mM Tris-HCl (pH 8.3 at 25°C) 4 mM MgCl 2 50 mM KCl – – – – 10 mM DTT 

T4 DNA Ligase 40 mM Tris-HCl (pH 7.8 at 25°C) 10 mM MgCl 2 – 0.5 mM ATP – – – 10 mM DTT 

If required for a specific application, dilute 

the enzyme with 1X reaction buffer for 

short-term use. 


276 



Molecular Cloning Products 

Selection Guide ................................................................................................................ 278 

Products ............................................................................................................................ 279 

CloneJET PCR Cloning Kit ............................................................................................ 279 

InsTAclone PCR Cloning Kit ......................................................................................... 281 

aLICator Ligation Independent Cloning (LIC) and Expression System ................... 282 

WELQut Protease ..........................................................................................................285 

Maxima H Minus Double-Stranded cDNA Synthesis Kit ...........................................286 

Fast DNA End Repair Kit .............................................................................................. 287 

Rapid DNA Ligation Kit ................................................................................................288 

TransformAid Bacterial Transformation Kit ............................................................... 289 

Phusion Site-Directed Mutagenesis Kit .....................................................................290 

Lambda DNA ................................................................................................................. 291 

Lambda DNA (dam – , dcm – ) ........................................................................................ 291 

X174 RF1 DNA ............................................................................................................ 291 

pBR322 DNA .................................................................................................................291 

pTZ19R DNA ..................................................................................................................292 

pUC57 DNA....................................................................................................................292 

pUC18 DNA ....................................................................................................................292 

pUC19 DNA ....................................................................................................................292 

Related Products by Application .................................................................................293 

Protocols and Recommendations ...............................................................................294 

3.1. DNA ligation .............................................................................................................294 

3.2. DNA blunting ............................................................................................................295 

3.3. Phosphorylation of DNA ............................................................................................295 

3.4. Dephosphorylation of DNA and RNA ..........................................................................295 

3.5. Removal of tags from recombinant proteins using WELQut Protease ...........................295 

3.6. Transformation .........................................................................................................296 

3.7. Cultivation of E.coli ....................................................................................................296 

3.8. Analysis of recombinant clones .................................................................................296 

Troubleshooting Guide ................................................................................................... 297 

Streamline your 

entire cloning workflow 

Learn more: 

www.thermoscientific.com/cloning 

Useful webtools: 

www.thermoscientific.com/REviewer 


278 

Selection Guide 

Applications Procedures Products to use Cat. # Page 

Digestion by restriction enzymes 

FastDigest Restriction Enzymes 


FDXXXX 

ERXXXX 

2 

77 

Generation of DNA 

fragments for cloning 

Preparation of cloning 

vector and insert for 

ligation 

Ligation of vector and 

insert DNA 


PCR & RT-PCR 

See complete portfolio for Thermo Scientific PCR & RT-PCR products 

at www.thermoscientific.com/pcr 

Maxima H Minus Double-Stranded cDNA Synthesis Kit K2561 286 

Double-stranded cDNA synthesis RNase H EN0201/2 266 

DNA Polymerase I EP0041/2 246 

GeneJET Plasmid Miniprep Kit K0502/3 304 

Purification of plasmid DNA 

GeneJET Plasmid Midiprep Kit K0481/2 305 

GeneJET Plasmid Maxiprep Kit K0491/2 306 

Dephosphorylation of cloning vector FastAP Thermosensitive Alkaline Phosphatase EF0651/2/4 242 

DNA end repair Fast DNA End Repair Kit K0771 287 

Blunting of DNA 

fragment ends 

3’- overhang removal 


S1 Nuclease 

EP0061/2 

EN0321 

249 

269 

5’- overhang fill in 


Klenow Fragment, exo 

EP0061/2 249 

– EP0421/2 248 

5’- overhang removal S1 Nuclease EN0321 269 

Phosphorylation of PCR products or 

synthetic DNA 

T4 Polynucleotide Kinase (T4 PNK) EK0031/2 243 

Purification from PCR or other reaction 

mixtures 

GeneJET PCR Purification Kit K0701/2 316 

GeneJET Gel Extraction Kit K0691/2 317 

Purification from agarose gel 

Silica Bead DNA Gel Extraction Kit K0513 318 

Agarase EO0461 319 

Blunt-end cloning CloneJET PCR Cloning Kit K1231/2 279 

TA cloning InsTAclone PCR Cloning Kit* K1213/4 281 

Ligation 

Rapid DNA Ligation Kit 

T4 DNA Ligase 

K1422/3 

EL001X 

288 

239 

Ligation Independent Cloning (LIC) aLICator LIC Cloning and Expression System 

K1241/51/61/ 

71/81/91 

282 

Transformation TransformAid Bacterial Transformation Kit K2710/1 289 

Protein expression in E.coli aLICator LIC Cloning and Expression System 

K1241/51/61/ 

71/81/91 

282 

Site-Directed Mutagenesis Phusion Site-Directed Mutagenesis Kit F-541 290 

pUC18 DNA SD0051 

Cloning vectors Closed circular plasmids 

pUC19 DNA 

pUC57 DNA 

SD0061 

SD0171 

292 

pTZ19R DNA SD0141 

pBR322 DNA SD0041 291 

Agarose 


TopVision Low Melting Point Agarose 

R0491/2 

R0801 

427 

Analysis of DNA 

Electrophoresis buffers 

10X TBE Buffer 

50X TAE Buffer 

B52 

B49 

371 

DNA ladders Wide range of Thermo Scientific DNA ladders SMXXXX 358 

X-Gal R0401/2/4 

Cultivation of E.coli Media additives 

X-Gal Solution, ready-to-use 

IPTG 

R0941 

R0391/2/3 

424 

IPTG Solution, ready-to-use R1171 

* not available in USA.

Products 

CloneJET PCR Cloning Kit 

#K1231 for 20 rxns 



Nucleotides p.408 


GeneJET Plasmid Miniprep Kit p.304 

GeneJET PCR Purification Kit p.316 

GeneJET Gel Extraction Kit p.317 

FastDigest Restriction Enzymes p.2 

pJET1.2 Sequencing Primers p.416 

Maxima H Minus Double-Stranded 

cDNA Synthesis Kit p.286 

Reference 

1. Michelsen, B.K., Anal. Biochem., 225, 172, 1995. 

Description 

The Thermo Scientific CloneJET PCR Cloning 

Kit is an advanced positive selection system 

for high efficiency cloning of PCR products 

generated with any thermostable DNA 

polymerase. Additionally, any other blunt or 

sticky-end DNA fragment can be cloned. 

The kit is ideal for phosphorylated or nonphosphorylated 

DNA fragments. Ligation into 

the included positive selection vector takes 

only 5 minutes and yields more than 99% 

recombinant clones. Blunt-end PCR products 

generated with a proofreading enzyme 

(e.g., Pfu or Phusion polymerase) are ligated 

directly into the cloning vector. PCR products 

generated either with non-proofreading DNA 

polymerases (e.g., Taq polymerase) or mixtures 

of DNA polymerases are blunted prior to 

ligation in 5 minutes with the thermostable 

DNA Blunting Enzyme provided with the kit. All 

common laboratory E.coli strains can be directly 

transformed with the ligation product. 

The kit contains a novel, ready-to-use positive 

selection cloning vector – pJET1.2/blunt. The 

vector contains a lethal restriction enzyme gene 

that is disrupted by ligation of a DNA insert 

into the cloning site. As a result, only bacterial 

cells with recombinant plasmids are able to 

form colonies. Recircularized pJET1.2/blunt 

vector molecules lacking an insert express a 

lethal restriction enzyme which kills the host 

E.coli cell after transformation. This positive 

selection drastically accelerates the process of 

colony screening and eliminates additional costs 

required for blue/white selection. 

For convenience in mapping and manipulation 

of the insert, the pJET1.2/blunt cloning 

vector multiple cloning site contains two BglII 

recognition sequences that flank the insertion 

site. 

In addition, the vector contains a T7 promoter 

for in vitro and in vivo transcription as well as 

sequencing of the insert. 

Table 3.1. Thermo Scientific CloneJET cloning flowchart. 

Procedure 

Blunt-end 

PCR 

product 

3’-dA tailed 

PCR 

product 

Blunting – 5 min 

Ligation 5 min 5 min 

Total time 5 min 10 min 

Features 

Fast – PCR cloning in only 5 minutes. 

Highest efficiency – >99% of positive clones. 

No cloning background – positive selection 

vector. 

Versatile – ideal for of cloning blunt-and 

sticky-end DNA fragments. 

Economical – no expensive blue/white 

screening. 

Applications 

Cloning of blunt-end or 3’-dA tailed PCR 

products up to 10 kb. 

Cloning of DNA fragments generated by 

restriction enzymes. 

Cloning of ds cDNA. 

cDNA library construction. 

Sequencing of cloned DNA. 

In vitro and in vivo transcription of cloned 

inserts from the T7 promoter. 

Components of the Kit 

 

 

 

 

 

 

 

 

 

Notes 

 

inactivate T4 DNA ligase by chloroform 

extraction or spin column purification, 

e.g., with GeneJET PCR Purification Kit 

(#K0701) (1). 


280 


pJET1.2/blunt vector map. 

Transformants/μg DNA 

2.5x10 5 

2x10 5 

1.5x10 5 

1x10 5 

5x10 4 

0 


CloneJET PCR 

cloning kit 

5 min 

Ligase-based blunt 

PCR cloning kit 

60 min 

Other vendors 

Topoisomerase-based 

blunt PCR cloning kit 

5 min 

Cloning of blunt-end PCR products: efficiency of different blunt-end PCR 

cloning systems with positive selection. A 976 bp blunt-end PCR product, 

generated with Pfu DNA Polymerase, was directly ligated into different positive 

selection blunt-end PCR cloning vectors according to the suppliers, protocols. Ligation 

mixtures (2 μl) were used to transform E.coli DH10B cells. Transformation efficiency of 

competent cells was 1x10 7 transformants per μg supercoiled DNA. 

Transformants/μg DNA 

5x10 5 

4x10 5 

3x10 5 

2x10 5 

1x10 5 

0 


CloneJET PCR 

cloning kit 

10 min 

InsTAclone PCR 

cloning kit 

60 min 

vendor A 

vendor B 

Topoisomerase- Ligase-based 

based TA cloning kit TA cloning kit 

5 min 60 min 

Cloning of 3’-dA tailed PCR products: efficiency of different PCR cloning 

strategies. A 976 bp 3’-dA tailed PCR product, generated with Taq DNA Polymerase, 

was ligated into pJET1.2/blunt and into different TA cloning vectors according to the 

suppliers, protocols. Ligation mixtures (2 μl) were used to transform E.coli DH10B 

cells. Transformation efficiency of competent cells was 1x10 7 transformants per μg 

supercoiled DNA.

InsTAclone PCR Cloning Kit 

(Not available in USA) 






IPTG p.424 

X-Gal p.424 

Sequencing Primers p.416 





PCR product cloning procedure. 

bla (Ap R ) 

pTZ57R/T 

2886 bp 

rep (pMB1) 

Restriction map of vector pTZ57R/T. 

f1 (IG) 

Description 

The Thermo Scientific InsTAclone PCR Cloning 

Kit is a TA system for direct one-step cloning 

of PCR products with 3’-dA overhangs (1) 

generated by 

Taq DNA Polymerase and other thermostable 

DNA polymerases which lack proofreading 

activity. The kit includes a ready-to-use cloning 

vector pTZ57R/T (see Restriction map below) 

constructed on the backbone of the pTZ57R 

vector. The 3’-ddT overhangs at both ends of 

the cloning site prevent recircularization of the 

vector during ligation, resulting in high cloning 

yields and low background. To increase the 

speed, convenience and efficiency of cloning 

experiment, the InsTAclone PCR Cloning Kit has 

been combined with the unique TransformAid 

Bacterial Transformation Kit. Ligation and 

preparation of competent cells is performed in 

parallel; therefore, it takes only one hour from 

the completion of PCR to cell plating. 

Our transformation protocol is often faster than 

the transformation of commercially available 

competent cells. 

lacZ 

ddT 

ddT 

P T7 

EcoRI 

Ecl136II 

SacI 

Acc65I 

KpnI 

Bsp68I 

Mva1269I 

Mph1103I 

XbaI 

ddT 

ddT 

BamHI 

Cfr9I 

Eco88I 

SmaI 

ApaI 

Bsp120I 

HincII 

SalI 

XmiI 

PstI 

AlfI 

Eco147I 

PaeI 

HindIII 

T7 promoter 

Features 

Fast – approximately one hour from PCR 

completion to cell plating. 

High efficiency – more than 90% of the 

recombinant clones contain the target DNA. 

One-step procedure – additional 

modifications of the PCR fragment are not 

required. 

Compatible with Taq, Tth, Tfl and other nonproofreading 

DNA polymerases, as well as 

with the Long PCR Enzyme Mix. 

Convenient pTZ57R/T cloning vector: 

– ready-to-use: linearized and 3’-ddT tailed, 

– MCS designed for easy mapping and 

manipulation of the cloned insert, 

– blue/white screening, 

– M13/pUC primer sites for sequencing or 

colony PCR screening, 

– T7 promoter for in vitro transcription of 

the insert. 

Applications 

TA cloning. 

Sequencing of cloned insert. 

In vitro transcription of insert DNA. 


 

 

 

 

 

 

 

 

 

 

 

Notes 

E.coli 

strains. E.coli cells are not included. 

Reference 

1. Clark, J.M., Novel non-templated nucleotide addition 

reactions catalyzed by prokaryotic and eukaryotic DNA 

polymerases, Nucl. Acids Res., 16 (20), 

9677-9686, 1988. 


282 

aLICator Ligation Independent Cloning (LIC) and Expression System 

aLICator LIC Cloning and Expression Kit 1 








aLICator LIC Cloning and Expression Set 1 





DNA Ladders pp.364-367 



WELQut Protease p.285 

IPTG p.424 



Description 

The Thermo Scientific aLICator LIC Cloning and 

Expression System is designed for fast and 

efficient ligation independent cloning and tight 

regulation of gene expression in E. coli. The 

pLATE bacterial expression vectors are designed 

for high levels of target protein expression in 

concert with minimal background (uninduced) 

expression, which permits expression of 

proteins that are toxic to E. coli cells. To 

streamline and facilitate the process of insert 

cloning into the expression vector, the aLICator 

system uses directional LIC cloning technology, 

a rapid and robust procedure that consistently 

provides high cloning efficiencies. 

The aLICator system consists of six kits based 

on the pLATE series of bacterial expression 

vectors. For proteins with a known preference 

for either the N- or C-terminal 6xHis-tag 

position, using the appropriate N- or C-terminal 

kit is recommended. When the protein 

structure and features are not well known, it is 

recommended to use a set, clone into all three 

vectors and determine the most compatible 

vector for further research. Following protein 

affinity purification, amino-terminal tags can be 

removed either via enterokinase (EK), DDDDK ^ 

or a novel WELQut (WQ), WELQ ^ cleavage sites 

that are located immediately N-terminal to the 

target protein. 

Table 3.2. Thermo Scientific aLICator cloning system overview. 

Features 

High efficiency robust directional LIC cloning. 

Tight control of gene expression. 

High yield expression. 

Versatile – tagged or untagged protein 

expression with tag removal option. 

Applications 

Directional PCR product cloning. 

Tightly regulated protein expression. 

Expression of toxic genes. 

Cat# aLICator LIC Cloning Kits Feature 

#K1241 aLICator LIC Cloning and Expression Kit 1 

#K1251 

#K1261 

#K1281 

#K1271 

#K1291 


(N-terminal His-tag/EK) 


(C-terminal His-tag) 


(N-terminal His-tag/WQ) 


(All-in-One/EK) 


(All-in-One/WQ) 

pLATE11 vector, untagged protein 

expression 

pLATE51 vector, N-terminal His-tag protein 

expression, enterokinase cleavage 

pLATE31 vector, C-terminal His-tag protein 

expression 

pLATE52 vector, N-terminal His-tag protein 

expression, WELQut cleavage 

pLATE11, pLATE51 and pLATE31 vectors, 

choice of untagged, N- or C-terminal His-tag 

protein expression, enterokinase cleavage 

pLATE11, pLATE52 and pLATE31 vectors, 

choice of untagged, N- or C-terminal Histag 

protein expression, WELQut cleavage

P T7 – T7 RNA polymerase promoter 

T rrnBT1-T2 – Transcription terminator 

lacO – lac operator 

RBS – Ribosome binding site 

EKS – Enterokinase recognition sequence 

WQS – WELQut protease recognition sequence 

P tet – P tet promoter 

T T7 – T7 terminator 

lacI – lac repressor 

rop – rop protein regulates plasmid copy number 

rep (pMB1) – Origin of replication (rep) 

of the pMB1 plasmid 

bla (Ap R ) – -lactamase gene 

6xHis – Polyhistidine tag 

pLATE vector elements. 

pLATE expression vectors use elements from bacteriophage T7 to control expression of heterologous genes in E.coli. The expression of the gene of interest is driven by 

a strong bacteriophage T7 promoter, which is specifically recognized by T7 RNA polymerase. To express the gene of interest, E.coli strains such as BL21 (DE3), 

HMS 174 (DE3) must be used, in which expression of T7 RNA polymerase gene is under the control of an inducible promoter such as lacUV5. 

After IPTG induction, T7 RNA polymerase is expressed within the cell, and starts transcription of genes under the T7 promoter. 


284 

Generation of sticky ends on the gene of interest with T4 DNA polymerase and dGTP. 

The LIC method uses T4 DNA polymerase to create specific 14 – 21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts (2). T4 DNA 

polymerase has two enzymatic activities: 3’ – 5’ exonuclease activity removes nucleotides from the 3’ ends of the DNA while 5’ – 3’ polymerase activity restores 

the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction mix, causing the exonuclease 

and polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand. After annealing, the LIC vector and insert are transformed 

into competent E.coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid. 

M 1 2 3 4 5 6 M N C M N C M N C M N C M 

Expression of a toxic gene. 

Restriction endonuclease Cfr9I R gene was cloned into 

pLATE expression vectors expressed in E.coli ER2566 

cells without expressed methylase. 

3 hours post-induction, bacterial cells were collected, 

normalized according to optical density and sonicated. 

1 μl of cell-free extract was assessed for Cfr9I 

restriction activity by its ability to cleave lambda DNA. 

M – GeneRuler High Range DNA Ladder (#SM1353), 

1 – Lambda DNA, 

2 – Lambda DNA / Cfr9I, 

3, 4, 5, 6 – cell-free extract with Cfr9I expressed within 

pLATE11, pLATE31, pLATE51 and pLATE52 

vectors respectively. 


0.5 μg Cat 0.5 μg Klenow 4 μg Cat 4 μg Klenow 

Functionality of C-terminal and N-terminal 6XHis-tags. 

M – PageRuler Prestained Protein Ladder, 

C – pLATE31 (C-terminal His-tag), 

N – pLATE51 (N-terminal His-tag), 

Cat – chloramphenicolacetyl transferase, 

Klenow – Klenow Fragment, exo – . 

References 

1. Studier F.W. and Moffatt B.A., Use of bacteriophage T7 RNA 

polymerase to direct selective high-level expression of cloned 

genes. J Mol Biol, 189:113-130, 1986. 

2. Aslanidis C., de Jong P. J., Ligation-independent cloning 

of PCR products (LIC-PCR). Nucleic Acids Res., 18(20): 

6069-6074, 1990. 

3. Rand, K.N., Crystal Violet can be used to Visualize DNA Bands 

during Gel Electrophoresis and to Improve Cloning Efficiency, 

Elsevier Trends Journals Technical Tips, Online, T40022, 1996. 

4. Adkins, S., Burmeister, M., Visualization of DNA in agarose 

gels and educational demonstrations, Anal Biochem., 240 (1), 

17-23, 1996.

WELQut Protease 

#EO0861 500 u (5 u/μl) 



(N-terminal His-tag/WQ) p.282 


(All-in-One/WQ) p.282 

Target 

protein 

Specific 

cleavage 

product 

WELQut 

Protease 

WELQut Protease Enterokinase 

Description 

The Thermo Scientific WELQut Protease is 

highly specific, recombinant serine protease 

of Staphylococcus aureus. It recognizes 

and precisely cleaves recombinant proteins 

containing an engineered recognition sequence* 

W- E- L- Q X (Trp, Glu, Leu, Gln, 

X can be any amino acid). The protease cleaves 

outside the recognition sequence without 

leaving additional amino acids bound to the 

target protein. 

The WELQut Protease is active in a broad 

temperature (4-30°C) and pH (pH 6.5-9.0) 

range and does not require specific buffers. 

In addition, this new protease has several 

procedural advantages – it is ideal for 

on-column proteolysis reactions and can be 

easily removed from reaction mixtures using its 

built-in His-tag. 

* This cleavage sequence is present in expression vector 

included into the Thermo Scientific aLICator LIC Cloning 

and Expression Kit 4 (N-terminal His-tag/WQ) available from 

Thermo Scientific (#K1281). 

M K 1 2 3 4 5 6 K 7 8 9 10 11 M 

Highly specific proteolysis of target protein performed by Thermo Scientific 

WELQut Protease vs. Enterokinase. 

Target protein (Klenow Fragment, exo – ), containing a WELQut Protease or 

Enterokinase recognition sequence) was treated with WELQut Protease or Enterokinase 

according to the recommendations provided by the suppliers. Various enzyme/ 

substrate ratios were tested and reaction products were analyzed in SDS-PAGE. 

M – PageRuler Prestained Protein Ladder (Thermo Scientific, #SM0671). 

K – Control - uncut target protein. 1-6 various WELQut/target protein amount ratios, u/μg. 

1 – 1:100, 2 – 1:50, 3 – 1:40, 4 – 1:20, 5 – 1:10, 6 – 1:5. 

7-11 various Enterokinase/target protein amount ratios, μg/μg. 

7 – 1:100, 8 – 1:40, 9 – 1:20, 10 – 1:10, 11 – 1:5. 

Non-specific cleavage 

products 

Features 

Cleaves outside WELQ recognition sequence, 

without leaving additional amino acids bound 

to the target protein. 

Highly specific to cognate recognition site, 

does not generate non-specific product 

bands, even after long incubation. 

Easy to remove from the reaction mixture 

using the built-in His-tag. 

Ideal for on-column proteolysis reactions. 

Applications 

Removal of N-terminal fusion tags from 

recombinant protein preparations. 


Each unit is defined as the amount of enzyme 

required to cleave 99% of 100 μg of a control 

protein in 16 h at 20°C. 

Enzyme activity is assayed in 100 μl 100 mM 

Tris-HCl (pH 8.0). 

Source 

Bacillus subtilis cells with a cloned gene of SplB 

protease from Staphylococcus aureus. 




Enzyme is supplied in: 10 mM Na 2HPO 4; 

1.8 mM KH 2PO 4, pH 7.3; 140 mM NaCl; 

2.7 mM KCl; 50% glycerol. 


» 3.5.1. Optimization of WELQut 

Protease cleavage p.295 

» 3.5.2. Cleavage of fusion proteins 

in solution p.296 

» 3.5.3. Cleavage of fusion proteins 

during affinity purification p.296 


286 

Maxima H Minus Double-Stranded cDNA Synthesis Kit 



GeneJET RNA Purification Kit p.307 

GeneJET Plant RNA Purification Kit p.309 

GeneJET Whole Blood RNA 

Purification Kit p.308 

CloneJET PCR Cloning Kit p.279 



DNA Ladders pp.364-367 

RNA Ladders p.386 


» 3.1.2. Blunt-end ligation p.294 


Description 

The Thermo Scientific Maxima H Minus 

Double-Stranded cDNA Synthesis Kit is an 

efficient system for synthesis of high quality 

double-stranded blunt end cDNA from total 

RNA or mRNA. The kit works at a wide range of 

starting RNA amounts (1-20 μg total RNA, 50 

ng-5 μg mRNA) and at elevated temperatures 

(50-60°C). First- and second-strand cDNA 

synthesis reactions are performed in the 

same tube without the need for intermediate 

purification steps. The kit contains premixed 

components to reduce the number of pipetting 

steps necessary to complete the procedure. 

This convenient one-tube format speeds up the 

synthesis procedure, reduces risk of pipeting 

error and maximizes cDNA recovery. 

The kit uses an advanced reverse transcriptase, 

Maxima H Minus, which was developed through 

in vitro evolution of M-MuLV RT for increased 

thermostability and speed. In addition, it lacks 

RNase H activity, resulting in greater cDNA 

yields and increased full-length cDNA. 

The kit is a complete solution. All necessary 

components are supplied within the kit: both 

oligo(dT) 18 and random hexamer primer primers, 

controls, RiboLock RNase Inhibitor, RNase I for 

residual RNA removal. 

Yield, % 

Features 

Efficient synthesis of full-length doublestranded 

cDNA. 

Fast – procedure completed in less than 

2 hours. 

Convenient – premixed components. 

Complete – includes all primers, controls and 

residual RNA removal reagents. 

Applications 

Full-length double-stranded blunt-end cDNA 

synthesis for cloning. 

Double-stranded cDNA library construction. 


 

 

 

 

 

 

 

18 Primer 

 

 

 

100 

90 


Vendor A 

Thermo 

Scientific vendor A vendor B 

80 

70 

60 

50 

40 

30 

20 

10 

Vendor B 

0 

M ss ds ss ds ss ds M 

1a 1b 

Efficient synthesis of double-stranded cDNA. 

20 μg of total Jurkat RNA was used as a template for oligo-dT primed first and second strand cDNA synthesis 

using the Maxima H Minus Double-Stranded cDNA Synthesis Kit and kits from other vendors, according 

to the manufacturers, instructions. Yields of purified reaction products were estimated using a NanoDrop 

spectrophotometer (calculated based on assumption that mRNA accounts for 5% of total RNA) (1a). One half 

of the each reaction product was analyzed on agarose gel (1b). 

M – ZipRuler Express DNA Ladder Mix (#SM1373) 

ss – single-stranded cDNA 

ds – double-stranded cDNA

Fast DNA End Repair Kit 









M C1 C2 T A B M 

Efficient ligation after DNA end repair with 

the Thermo Scientific Fast DNA End Repair Kit. 

A 130 bp PCR product with 3’- dA overhangs was 

end-repaired using the Fast DNA End Repair Kit and 

kits from other vendors according to the suppliers’, 

recommendations, and subsequently ligated with T4 

DNA Ligase (#EL0014). Ligation conditions: 200 ng of 

130 bp PCR product ligated with 5u T4 DNA Ligase 

in 1X T4 DNA Ligase Buffer with 5% PEG 4000 for 

1h at 22°C. 

M – GeneRuler 1 kb Plus DNA Ladder (#SM1333), 

C1 – PCR product, 

C2 – PCR product after ligation, 

T – PCR product after end repair using Fast DNA End 

Repair Kit (20°C, 5 min) and ligation, 

A – PCR product after end repair using Vendor A’s kit 

(20°C, 30 min) and ligation, 

B – PCR product after end repair using Vendor B’s kit 

(RT, 45 min; 70°C, 10 min) and ligation. 

Description 

The Thermo Scientific Fast DNA End Repair 

Kit is used for blunting and phosphorylation 

of DNA ends in just 5 minutes for subsequent 

use in blunt-end ligation. During the DNA end 

repair reaction, fragmented DNA is converted 

into blunt-end DNA containing 5’-phosphate 

and 3’-hydroxyl groups. The 5’3’ polymerase 

activity of the End Repair Enzyme Mix fills-in 

5’ protruded DNA ends while its 3’5’ 

exonuclease activity removes 3’-overhangs. 

T4 polynucleotide Kinase (PNK) adds 

5’-phosphates to ends of unphosphorylated 

DNA fragments such as PCR products. 

All components of the kit contain premixed 

reagents to reduce pipetting steps and provide 

convenience. The End Repair Enzyme Mix 

contains an optimized mixture of T4 DNA 

Polymerase and Klenow Fragment to achieve 

highly effective blunting of fragmented DNA, 

and T4 PNK for efficient phosphorylation of 

DNA ends. The 10X End Repair Reaction Mix 

contains an optimized reaction buffer, 

ATP and dNTPs. 

Features 

Efficient – blunting and phosphorylation of 

0.5-5 μg DNA. 

Fast – reaction is completed in 5 minutes. 

Convenient – reaction components are 

premixed to reduce pipetting steps. 

Applications 

Blunting and phosphorylation of doublestranded 

DNA (nebulized DNA, sonicated 

DNA, restriction enzyme digested DNA, cDNA, 

PCR products). 


 

 

 

5’-overhangs 3’-overhangs Blunt PCR product PCR product with 3’-dA overhangs 

dA 

dA 5’ 

5’ 

5’ 

5’ 

P 

P 

P 

Blunting and phosphorylation of different types of DNA ends 

P 

P 

P 


» 3.1.2. Blunt-end ligation p.294 

P 

P 


288 

Rapid DNA Ligation Kit 





Cloning Vectors p.292 

IPTG p.424 

X-Gal p.424 



FastAP Thermostabile Alkaline Phosphatase p.242 





Fast DNA End Repair Kit p.287 

Maxima H Minus Double Strand cDNA Kit p.286 



Description 

The Thermo Scientific Rapid DNA Ligation Kit 

allows fast sticky-end or blunt-end DNA ligation 

in only 5 min at room temperature. 

The kit contains T4 DNA Ligase and a specially 

formulated 5X rapid ligation buffer optimized 

for fast and efficient DNA ligation. Fast ligation 

efficiency is equal to that obtained with T4 

DNA Ligase in a standard 1 hour ligation. The 

ligation reaction mixture can be used directly for 

bacterial transformation with the TransformAid 

Bacterial Transformation Kit or with other 

conventional transformation procedures. 

Features 

Fast – ligation of either sticky- or blunt-end 

DNA in only 5 minutes. 

Convenient – reaction mixture can be used 

directly for bacterial transformation. 

Transformants per μg DNA 

10 7 

10 6 

10 5 

10 4 

10 3 

sticky ends 

blunt ends 

0 5 10 15 20 25 30 

Time, min 

Ligation of sticky- and blunt-end DNA 

fragments with the Thermo Scientific 

Rapid DNA Ligation Kit. 

Blunt-end ligation: pUC19 DNA/SmaI dephosphorylated 

vector was ligated with DNA/PvuII fragment (2.3 kb). 

Sticky-end ligation: pUC19 DNA/PstI dephosphorylated 

vector was ligated with DNA/PstI fragment (2.1 kb). 

The efficiency of ligation was determined by 

transformation of E.coli XL1-Blue cells. 

Applications 

Routine cloning experiments. 

Blunt-end cloning. 

Library construction. 

TA cloning. 


 

 

 

 

Notes 

 




(#K0701) (1). 

Reference 

1. Michelsen, B.K., Anal. Biochem., 225,172, 1995.

TransformAid Bacterial Transformation Kit 

#K2710 for 20 transformations 

#K2711 for 40 transformations 



T4 DNA Ligase 

FastAP Thermostabile Alkaline 

p.239 





Cloning Vectors p.292 

IPTG p.424 

X-Gal p.424 


From Overnight Bacterial Culture 

Add an aliquot of overnight 

culture to C-medium 

Incubate 20 min at 37°C 

Aliquot, centrifuge 1 min 

Resuspend in T-solution 

Incubate 5 min on ice 

Centrifuge 1 min 


incubate 5 min on ice, aliquot 

Mix with DNA 

transform 5 min 

Plate immediately 

Description 

The Thermo Scientific TransformAid Bacterial 

Transformation Kit uses a novel method for 

rapid preparation of chemically competent 

E.coli cells from overnight bacterial cultures 

or bacterial colonies. The key component of 

this system is the unique T-solution, which 

produces competent cells in a few easy steps. 

The quick and convenient procedure guarantees 

transformation efficiencies of more than 107 transformants per μg of plasmid DNA, ideal for 

routine cloning experiments. The option to use 

bacterial colonies for preparation of competent 

cells adds flexibility to any cloning experiment. 

The TransformAid Bacterial Transformation Kit 

can be used with most E.coli strains commonly 

used for cloning. 

Features 

Efficient – more than 107 transformants 

per μg of plasmid DNA. 

Practical – E.coli competent cells can be 

prepared from an overnight culture or from 

bacterial colonies. 

Fast – less than 1 hour from the overnight 

bacterial culture to cell plating. Starting from 

bacterial colonies, the procedure requires 

only 2.5 hours. 

Easy – all procedures are performed on 

ice on your bench. Brief centrifugations are 

carried out in standard microcentrifuges. 

From Bacterial Colonies 

Add a few colonies to 

C-medium 

Incubate 2 hours at 37°C 

Aliquot, centrifuge 1 min 


Incubate 5 min on ice 



incubate 5 min on ice, aliquot 

Mix with DNA 

transform 5 min 

Plate immediately 

Total time: 50 min 

Total time: 2.5 hours 

Transformation workflow with the Thermo Scientific TransformAid Bacterial Transformation Kit. 

Applications 

Routine cloning experiments. 

Blunt-end cloning. 

TA cloning. 


 

 

 

 

Notes 

 

Bacterial Transformation Kit are suitable for 

direct use only. Freezing down and storage 

at -70°C is not recommended. 


» 3.7. Cultivation of E.coli p.296 


290 

Phusion Site-Directed Mutagenesis Kit 

#F-541 for 20 rxns incl. 

10 control rxns 


IPTG p.424 

X-Gal p.424 


POINT MUTATION DELETION INSERTION OPTION 1 

R 

5'P 

Mu t 

Linear amplified target plasmid 

with desired mutation 

The Phusion Site-Directed Mutagenesis protocol 


X 

5'P 

Target 

plasmid 

F 

R 

Del 

5'P 

5'P 

Target 

plasmid 

5'P 

X 

F 

X 

Mutated target 

plasmid 

Description 

The Thermo Scientific Phusion Site-Directed 

Mutagenesis Kit is a versatile and efficient 

tool for introducing point mutations, insertions 

or deletions in any type of plasmid DNA. With 

this kit, the entire plasmid is amplified using 

phosphorylated primers that introduce the 

desired changes. The amplified linear PCR 

product, containing the desired mutation, is 

circularized in a 5-minute ligation reaction. The 

resulting plasmid then can be transformed into 

competent E.coli cells. 

5'P 

R 

Ins 

5'P 

Target 

plasmid 

5'P 

F 

INSERTION OPTION 2 

R 

5'P Ins 

5'P 

Target 

plasmid 

Step 1. 

Amplification of target plasmid 

with two phosphorylated primers. 

Step 2. 

Plasmid circularization 

by ligation. 

Step 3. 

Transformation into E. coli. 

F 

Features 

Robust and reliable exponential amplification 

method. 

No specific requirements for vectors, 

restriction sites or methylation status of the 

target plasmid. 

No need to destroy the starting template in a 

separate step. 

High fidelity Phusion Hot Start II DNA 

Polymerase minimizes unwanted secondary 

mutations. 

Amplification of large plasmids up to 10 kb. 

Hot start modification of the polymerase 

prevents amplification of non-specific 

products and unwanted degradation of 

primers prior to first cycle of PCR. 

Compatible with all strains of competent 

E.coli cells. 

Applications 

Generating point mutations, insertions and 

deletions in plasmid DNA. 

Storage 

Store at -20°C. 


 

 

 

 

 

 

 

Notes 

 




(#K0701) (1). 

Reference 

1. Catic, et al., Sequence and structure evolved separately in a 

ribosomal ubiquitin variant., EMBO J. 26, 3474-3483, 2007.

Lambda DNA 

#SD0011 500 μg 

(0.3 μg/μl) 


10 mM Tris-HCl (pH 7.6) and 1 mM EDTA. 

Lambda DNA (dam – , dcm – ) 

#SD0021 500 μg 

(0.3 μg/μl) 



X174 RF1 DNA 

#SD0031 50 μg 

(0.5 μg/μl) 



pBR322 DNA 

#SD0041 100 μg 

(0.5 μg/μl) 



Description 

Linear double-stranded lambda bacteriophage 

(cI857 Sam7) DNA, 48502 base pairs with a 

molecular weight of 31.5 x 10 6 Da. Isolated from 

a heat-inducible lysogenic E.coli W3110 strain. 

For map and features see p.434. 

Description 

Linear double-stranded Dam and Dcm 

methylation-free lambda bacteriophage 

(cI 857 Sam7) DNA, 48502 base pairs with a 

molecular weight of 31.5 x 10 6 Da. Isolated 

from a heat-inducible lysogenic E.coli GM2163 

strain. For map and features see p.434. 

Description 

Double-stranded closed circular plasmid, 

5386 base pairs with a molecular weight of 

3.50 x 10 6 Da. Isolated from E.coli HF4704 

infected with X174 am3. For map and 

features see p.435. 

Description 

Double-stranded closed circular medium copy 

plasmid, 4361 base pairs with a molecular 

weight of 2.83 x 10 6 Da. Isolated from 

E.coli (dam + , dcm + ). For map and features 

see p.439. 

Applications 

Activity and specificity assays of restriction 

enzymes. 

Preparation of DNA molecular weight standards. 

Cloning. 

Applications 

Activity and specificity assays of restriction 

enzymes sensitive to dam or 

dcm methylation. 

Preparation of DNA molecular 

weight standards. 

Cloning. 

Features 

Purified by chromatography using proprietary 

technology. 

More than 90% in the supercoiled form. 

Application 

Preparation of DNA molecular weight 

standards. 

Features 


technology. 


Applications 

Cloning. 

Preparation of DNA molecular weight 

standards. 


292 

pTZ19R DNA 

#SD0141 50 μg 

(0.5 μg/μl) 



pUC57 DNA 

#SD0171 50 μg 

(0.5 μg/μl) 



pUC18 DNA 

#SD0051 50 μg 

(0.5 μg/μl) 



pUC19 DNA 

#SD0061 50 μg 

(0.5 μg/μl) 




Description 

Double-stranded closed circular high copy 

phagemid, 2862 base pairs with a molecular 

weight of 1.86 x 10 6 Da. Isolated from E.coli 

(dam + , dcm + ). For map and features see p.445. 

Description 





Description 





Description 





Features 


technology. 


Applications 

Cloning. 

Sequencing of insert DNA. 

In vitro transcription. 

Features 


technology. 


Applications 

Cloning. 


Generation of nested deletions with ExoIII. 

Features 


technology. 


Applications 

Cloning. 


Preparation of DNA molecular weight standards. 

Features 


technology. 


Applications 

Cloning. 

Sequencing of insert DNA.

Related Products by Application 

Dephosphorylation & phosphorylation Cat. # Size Supplied with Applications Page 


Phosphatase, 1 u/μl 

T4 Polynucleotide Kinase 

(T4 PNK), 10 u/μl 

EF0651 1000 u 10X FastAP Buffer 2x1.5 ml 

 

EF0652 5x1000 u 10X FastAP Buffer 10x1.5 ml 

cloning vector DNA during ligation. 

EF0654 300 u 10X FastAP Buffer 1.5 ml 

EK0031 500 u 

EK0032 2500 u 




10X Reaction Buffer A 2 ml 



 

products prior to ligation into 

dephosphorylated vector DNA. 

 

linkers and DNA or RNA prior to 

ligation. 


» 3.3. Phosphorylation of DNA 

» 3.4.2. Fast simultaneous plasmid vector linearization and dephosphorylation 

» 3.4.1. Dephosphorylation of DNA 5’-termini 

DNA blunting Cat. # Size Supplied with Applications Page 

T4 DNA Polymerase, 5 u/μl 

Klenow Fragment, exo – , 5 u/μl 

EP0061 100 u 10X Reaction Buffer 0.35 ml 

of 3’-overhangs or fill in of 

5’-overhangs. 

EP0062 500 u 10X Reaction Buffer 2x1 ml 

PCR products. 

EP0421 300 u 10X Reaction Buffer 1 ml 

EP0422 1500 u 10X Reaction Buffer 5x1 ml 5’-overhangs. 

S1 Nuclease, 100 u/μl EN0321 10000 u 5X Reaction Buffer 2x1 ml 

 

3’, 5’-overhangs. 

 

DNA fragments (in conjunction with 

Exonuclease III). 

242 

243 

p.295 

p.295 

p.295 


» 3.2.1. Blunting of 5’- or 3’-overhangs with T4 DNA Polymerase p.295 

» 3.2.2. Fill-in of 5’-overhangs with Klenow Fragment p.295 

» 3.2.3. Removal of 3’- and 5’-overhangs with S1 Nuclease p.295 

Cultivation of E.coli Cat. # Size Applications Page 

IPTG Solution, ready-to-use, 100 mM R1171 10x1.5 ml 

R0391 1 g 

IPTG, dioxane-free 

R0392 5 g 

R0393 5x5 g 

X-Gal Solution, ready-to-use, 20 mg/ml R0941 10 ml 

X-Gal 

Reference 

1. Sambrook, J., Russell, D.W., Molecular Cloning: A 

Laboratory Manual, the Third edition, Cold Spring Harbor 

Laboratory Press, Cold Spring Harbor, New York, 1.124- 

1.125, A1.27, 2001. 

R0401 0.5 g 

R0402 2x1 g 

R0404 1 g 

Blue/white colony screening, distinguishing recombinant (white) from nonrecombinant 

(blue) colonies (1). 


» 3.7.2. Preparation of X-Gal/IPTG LB agar plates for blue/white colony screening p.296 

249 

248 

269 

424 


294 


3.1. DNA ligation 

3.1.1. Sticky-end ligation 


Linear vector DNA 20-100 ng 

Insert DNA 

1:1 to 5:1 

molar ratio over vector 

10X T4 DNA Ligase Buffer 2 μl 

T4 DNA ligase 1 u 



2. Incubate 10 min at 22°C. 

3. Use up to 5 μl of the mixture to transform 

50 μl of chemically competent cells or 

0.5-1 μl per 50 μl of electrocompetent cells. 

Note 

The electrotransformation efficiency may be improved by: 

– heat inactivation of T4 DNA ligase at 65°C for 10 min or 

at 70°C for 5 min, 

– purification of DNA using the GeneJET PCR Purification 

Kit (#K0701) or by chloroform extraction. 

The overall number of transformants may be increased by 

extending the reaction time to 1 hour. 

If more than 2 u of T4 DNA ligase is used in a 20 μl 

reaction mixture, it is necessary to purify the DNA 

by spin column or chloroform extraction before 

electrotransformation. 

3.1.2. Blunt-end ligation 


Linear vector DNA 20-100 ng 

Insert DNA 

1:1 to 5:1 

molar ratio over vector 


50% PEG 4000 solution 2 μl 




2. Incubate 1 hour at 22°C. 


50 μl of chemically competent cells. 

Purify DNA for electrotransformation using 

the GeneJET PCR Purification Kit (#K0701) 

or by cloroform extraction. Use 1-2 μl of DNA 

solution per 50 μl of electrocompetent cells. 

3.1.3. Self-circularization of linear DNA 


Linear DNA 10-50 ng 





2. Mix thoroughly, spin briefly and incubate 

10 min at 22°C. 


50 μl of chemically competent cells and 

0.5-1 μl per 50 μl of electrocompetent cells. 


Note 

The electrotransformation efficiency may be improved by: 

– heat inactivation of T4 DNA ligase at 65°C for 10 min or 

at 70°C for 5 min, 

– purification of DNA using the GeneJET PCR Purification 

Kit (#K0701) or by chloroform extraction. 

The overall number of transformants may be increased by 

extending the reaction time to 1 hour. 

Important Notes 

 

in the rection mixture. 

 

in agarose gels. To avoid this, incubate samples with 

6X Loading Dye & SDS Solution (#R1151) at 65°C 

for 10 min and chill on ice prior to loading. 

 

reaction mixture should not exceed 10% of the competent 

cell volume. 

3.1.4. Linker ligation 

Double-stranded oligonucleotide linkers are 

often used to generate overhangs not found in 

the insert. Linkers normally contain restriction 

enzyme recognition sequences and are digested 

after ligation to generate overhangs compatible 

with cloning vectors. Alternatively, linkers may 

have overhangs which are ready for ligation 

with a cloning vector and do not require further 

manipulation following ligation. 


Linear DNA 100-500 ng 

Phosphorylated linkers 1-2 μg 


50% PEG 4000 solution 2 μl 




2. Mix thoroughly, spin briefly and incubate for 

1 hour at 22°C. 

3. Heat inactivate at 65°C for 10 min or at 70°C 

for 5 min. 

Note 

T4 DNA Ligase is active in PCR and restriction digestion 

buffers (see table below). Therefore, linker ligation reactions 

can be performed in the restriction enzyme buffer optimal 

for the subsequent digestion. In this case, the ligation 

reaction should be supplemented with ATP to a final 

concentration of 0.5 mM. After inactivation of the T4 DNA 

Ligase, add the restriction enzyme directly to the reaction 

mixture and incubate according to the digestion protocol. 

Buffers 

Activity in PCR and restriction digestion buffers 

Activity*, % 

PCR and RT buffers 75 

Reaction buffers for 

restriction enzymes 

FastDigest, 1X Tango, 

2X Tango, B, G, O, R, 

KpnI, BamHI, EcoRI 

75-100 

Ecl136II, SacI 50 

* activity of T4 DNA Ligase in various buffers supplemented with 0.5 mM ATP. 

3.1.5. Analysis of ligation products by 

agarose gel electrophoresis 

Ligation efficiency can be assessed by agarose 

gel electrophoresis of ligation reaction products. 

For sample loading, use of SDS-supplemented 

loading dye; e.g., 6X DNA Loading Dye & SDS 

Solution (#R1151) is recommended to eliminate 

band shift due to T4 DNA ligase binding to DNA 

(see Fig. below). 

1. Prepare the loading mixture: 

Ligation reaction product 10 μl 

6X DNA Loading Dye & SDS Solution (#R1151) 2 μl 

2. Heat the sample for 10 min at 65°C or for 

5 min at 70°C, chill on ice and load. 

1 2 3 M 

Analysis of ligation reaction products on an 

agarose gel. 

400 ng of vector and insert in total were used. Standard 

ligation experiments normally use less DNA; therefore, 

bands on a gel may appear at lower intensity. 

M – GeneRuler DNA Ladder Mix (#SM0331). 

1 – Mixture of DNA insert and vector in T4 DNA 

Ligase Buffer. 

2 – Mixture of DNA insert and vector after the ligation 

sample loaded with 6X DNA Loading Dye (#R0611). 

3 – Mixture of DNA insert and vector after the ligation 

sample loaded with 6X DNA Loading Dye & SDS 

Solution (#R1141). 

Interpretation of results 

Appearance of higher molecular weight 

bands and decreased intensity of the vector 

and insert bands indicate successful ligation. 

 

indicates unsuccessful ligation. 

3.1.6. Control reaction for T4 DNA 

Ligase activity 

Unsuccessful ligation may be the result of inactive 

ligase or inhibition of the ligation reaction by 

impurities in sample DNA. To assess the activity 

of T4 DNA Ligase we recommend performing a 

ligation reaction with control DNA; e.g., Lambda 

DNA/HindIII DNA Marker (#SM0101). 

1. Prepare the control ligation mixture: 

Lambda DNA/HindIII DNA Marker (#SM0101) 1 μl (0.5 μg) 


T4 DNA Ligase 1 u 


Total volume 20 μl

2. Mix thoroughly, spin briefly and incubate at 


3. Prepare the loading mixture: 

Ligation reaction product 10 μl 

6X DNA Loading Dye & SDS Solution (#R1151) 2 μl 

4. Heat the sample for 10 min at 65°C or for 

5 min at 70°C, chill on ice and load. 

1 2 

Evaluation of T4 DNA Ligase activity in control 

experiment. 

1 – Lambda DNA/HindIII fragments, unligated. 

2 – Lambda DNA/HindIII after ligation reaction. 

Interpretation of results 

Appearance of higher molecular weight bands 

and decreased intensity of the lower molecular 

weight bands indicates active ligase. 

 

indicates inactive ligase. 

3.2. DNA blunting 

3.2.1. Blunting of 5’- or 3’-overhangs 

with T4 DNA Polymerase 

T4 DNA Polymerase fills-in 5’-overhangs and 

removes 3’-overhangs. 


5X reaction buffer for T4 DNA Polymerase 4 μl 

Linear DNA or PCR product 1 μg 

dNTP Mix, 2 mM each (#R0241) 

1 μl (0.1 mM final 

concentration) 

T4 DNA Polymerase (#EP0061) 0.2 μl (1 u) 



2. Mix thoroughly, spin briefly and incubate at 11°C 

for 20 min or at room temperature for 5 min. 

3. Stop the reaction by heating at 75°C for 10 min. 

3.2.2. Fill-in of 5’-overhangs with 



Linear DNA 10-15 μl (0.1-4 μg) 

10X reaction buffer for Klenow Fragment 2 μl 

dNTP Mix, 2 mM each (#R0241) 0.5 μl (0.05 mM 

final concentration) 

Klenow Fragment, exo – (#EP0421) 0.1-0.5 μl (1-5 u) 





3. Stop the reaction by heating at 75°C for 10 min. 

3.2.3. Removal of 3’- and 5’-overhangs 

with S1 Nuclease 

S1 Nuclease removes 3’ and 5’ single-stranded 

DNA overhangs and hairpin loops. The activity 

of S1 Nuclease is substrate-dependent and the 

optimal enzyme and DNA amounts for successful 

blunting should be determined experimentally. 


DNA ~1 μg 

5X reaction buffer for S1 Nuclease 6 μl 

S1 Nuclease (#EN0321) 0.1 μl (10 u) 



2. Incubate the mixture at room temperature 

for 30 min. 

3. Stop the reaction by adding 2 μl of 0.5 M 

EDTA and heating at 70°C for 10 min. 

Note 

The S1 Nuclease can be diluted with 1X reaction buffer 

immediately prior to use. 

3.3. Phosphorylation of DNA 


Linear dsDNA or 

Oligonucleotide 

10X reaction buffer A for 

T4 Polynucleotide Kinase 

1-20 pmol of 5’-termini 

10-50 pmol 

2 μl 

ATP, 10 mM* 2 μl 

T4 Polynucleotide Kinase (#EK0031) 1 μl (10 u) 



* Prepare 10 mM ATP solution by combining 10 μl of 100 mM ATP 

solution (#R0441) and 90 μl of Water, nuclease-free. 



3. Heat at 75°C for 10 min. 

Note 

See Appendix on p.428 or visit 

www.thermoscientific.com/reviewer for molar calculations. 

3.4. Dephosphorylation of DNA 

and RNA 

3.4.1. Dephosphorylation of 

DNA and RNA 

This protocol is suitable for removal of 

3’- and 5’-phosphate groups from DNA and 

RNA. The protocol below is an example for 

dephosphorylation of ~3 kb linear vector DNA. 


Linear DNA (~3 kb plasmid) 

1 μg 

(~1 pmol termini) 

10X reaction buffer 2 μl 


Phosphatase (#EF0651) 

1 μl (1 u) 




37°C 10 min. 

3. Stop reaction by heating: 75°C for 5 min. 

Note 

For efficient dephosphorylation, plasmid DNA should be free 

of RNA and genomic DNA. 

3.4.2. Fast simultaneous plasmid vector 

linearization and dephosphorylation 

1. Prepare the following reaction mixture containing: 

Plasmid DNA 1 μg 

10X FastDigest Buffer 2 μl 

FastDigest Restriction Enzyme 1 μl 


(#EF0651) 

1 μl 





3. Stop the reaction by heating at 65°C for 

15 min or at 80°C for 20 min (if restriction 

enzyme is not inactivated at 65°C). 

Note 

For FastDigest SphI (PaeI) (#FD0601), simultaneous digestion 

and dephosphorylation is not recommended. Perform digestion, 

spin column purification and then dephosphorylation. 

3.5. Removal of tags from 

recombinant proteins using 

WELQut Protease 

3.5.1. Optimization of WELQut Protease 

cleavage 

Accessibility of the cleavage site, the adjacent 

amino acid sequence, and the degree of protein 

aggregation-all affect the cleavage efficiency. 

Optimal cleavage conditions must be determined 

individually for each protein to be cleaved. We 

recommend testing several enzyme/protein of 

interest ratios, concentrations, temperatures 

(4° to 30°C) or incubation time (1 to 16 h) to 

optimize the efficiency of cleavage. 

Optimization of the cleavage conditions should 

be performed in small-scale reactions using the 

following protocol as a starting point. 

1. Prepare samples: 

Component Amount 

Target protein 50 μg 

WELQut Protease* 0.5 u, 1 u, 2 u,10 u 

1x Reaction Buffer** Adjust to 50 μl 

Total 50 μl 

* Enzyme/protein amount ratios are 1:100, 1:50, 1:25, 1:5 (u/μg). 

For all recommended enzyme/protein ratios except 1:5 (u/μg), use 

10x diluted WELQut Protease. Dilute protease in the reaction buffer to 

the final concentration of 0.5 u/μl. 

** We recommend using 10-100mM Tris-HCl, pH 8.0 as the reaction 

buffer. 

2. Incubate at 15-30°C temperature. Take 8 μl 

aliquot from each reaction after 1, 3, 6, and 

16 h (or overnight). Follow standard protocol 

for preparation of protein samples prior 

SDS-PAGE analysis. 

Note 

For cleavage reactions longer than 16 h, incubation 

temperatures higher than 20°C are not recommended. 

3. Analyze the efficiency of cleavage in each 

sample by SDS-PAGE. 

Once optimal cleavage conditions have 

been found, the reaction can be scaled up 

proportionally for cleavage of fusion proteins 

in solution, in batch or on-column formats. 


296 

3.5.2. Cleavage of fusion proteins in 

solution 

1. We recommend using 10-100 mM Tris-HCl 

(pH 8.0), 10-100 mM Na3PO4 (pH 7.4) or 

10-20 mM K3PO4 (pH 7.4) as 1x buffer for the 

cleavage reaction. 

Note 

If target protein will be downstream purified using affinity 

chromatography, add NaCl and Imidazole to the reaction buffer 

at 50 mM and 5-20 mM final concentration, respectively. 

2. Add WELQut Protease to the fusion protein at 

an optimized protease/protein ratio. 

3. Incubate at 15-30°C temperature for the 

optimal time. 

Note 

If target protein is labile or cleavage reaction lasts longer than 

16 h, it should be performed in lower temperatures (4-20°C). 

4. Optional: removal of WELQut protease. 

Use the IMAC resins for post-cleavage 

purification of target protein according to 

the manufacturer’s instructions. In the IMAC 

column purification format, the protein of 

interest is eluted with the flow-through, while 

WELQut protease and the cleaved affinity tag 

remain bound to the resin. 

3.5.3. Cleavage of fusion proteins 

during affinity purification 

WELQut Protease can be used for hydrolysis of fusion 

proteins during affinity purification procedures. The 

protein of interest will be cleaved while it is still bound 

to the resin and further eluted while leaving the affinity 

tag and WELQut Protease, which contains built-in 6x 

His-tag, bound to the resin. 

On-column cleavage needs to be optimized for each 

fusion protein with respect to the amount of protease 

used and time required for cleavage (see protocol A). 

1. Evaluate the amount of target protein to be 

cleaved by SDS-PAGE. 

2. Bind the fusion protein to the metal-affinity 

resin of choice and wash according to the 

manufacturer’s instructions. 

3. Equilibrate the resin/column 2 times with 2-4 

resin volume of 50-100 mM Tris-HCl, 50 mM 

NaCl, 5-20 mM imidazole (pH 8.0), or buffer 

with 50-100 mM Na3PO4, 50 mM NaCl and 

5-20 mM imidazole (pH 7.4) or buffer with 

10-20 mM K3PO4, 50 mM NaCl, 5-20 mM 

imidazole (pH 7.4). 

4. Prepare mix of WELQut protease and the buffer 

used for resin/column equilibration in previous 

step. Use a protease/recombinant protein ratio 

determined in cleavage optimization experiment. 

Load the prepared WELQut protease mix 

on the column. 

Note 

If optimal protease/protein ratio was not determined, we 

recommend using 1:20 or 1:10 u/μg of WELQut to target 

protein ratio. 

5. Incubate at 15-30°C temperature for the 

optimal time. 

Note 

If optimal cleavage reaction time was not determined, we 

recommend 16 h (or overnight) incubation at 4-20°C. 

6. Collect eluate containing the protein of interest. 

WELQut Protease remains bound to the resin. 


7. Optional: Additional amount of equilibration buffer 

(0.5-1.0 settled resin volume) containing 0.2-0.5 M 

NaCl and 5-20 mM imidazole, may be used to wash 

the residual amount of protein of interest from the 

resin/column. Collect this diluted sample separately. 

8. Recommended: Remove the residual WELQut 

protease by running the eluate containing protein 

of interest through the fresh IMAC sorbent. Collect 

the flow-through. 

3.6. Transformation 

The number of transformants on the plates directly 

depends on the transformation efficiency of the 

competent cells. For successful cloning, competent 

E.coli cells should have an efficiency of at least 1x10 6 

transformants per μg supercoiled plasmid DNA. To 

check the efficiency, prepare a control transformation 

with 0.1 ng of a supercoiled vector DNA; e.g., pUC19 

DNA (#SD0061). 

Calculation of transformation efficiency 

For 500 colonies (cfu) on a control plate, the 

transformation efficiency is calculated as follows: 

500 cfu/0.1 ng of control DNA plated X 1000 = 

5x10 6 cfu/μg DNA. 

Transformation tips 

When using commercial competent cells, follow the 

recommendations from the supplier. 

Use up to 5 μl of the ligation mixture per 50 μl of 

chemically competent cells. 

For transformation by electroporation, extract 

the ligation reaction mixture with chloroform or a 

commercial spin column. Use 1 μl of purified ligation 

mixture to transform 50 μl of electrocompetent cells. 

Ligation efficiency often decreases as the insert 

size increases. Therefore, for vector containing large 

inserts (>5 kb) we recommend transformation by 

electroporation as it yields the highest transformation 

efficiencies (10 9 cfu/μg supercoiled DNA). 

3.7. Cultivation of E.coli 

3.7.1. Preparation of E.coli culture 

glycerol stocks 

Transformation efficiency and subsequent analysis of 

recombinant plasmids depends on the quality of the 

E.coli strains used in laboratory. Proper storage is vital 

to ensure competent cells retain high transformation 

efficiency. 

Agar plates are only suitable for short term 4°C 

storage. A glycerol stock kept at -70°C is the ideal 

way to store bacterial strains. 

1. When a lyophilized bacterial strain is purchased, 

a small portion of the powder is transferred with 

a sterile pipette tip or inoculating loop to liquid LB 

medium and incubated for 2-8 hours in a shaker. 

2. A drop of liquid culture is spread on a selective plate 

and propagated overnight at 37°C to check for 

presence of selective markers. 

3. Select a single colony and grow overnight. 

4. Add 180 μl of 87% sterile glycerol to a 2 ml 

screw-cap culture vial. 

5. Add 820 μl of liquid E.coli culture to vial, mix well, 

freeze in liquid nitrogen and store at -70°C. 

When recovering bacteria from a glycerol stock, it 

is recommended to check for selective markers by 

streaking an aliquot on a selective plate. 

3.7.2. Preparation of X-Gal/IPTG LB agar 

plates for blue/white colony screening 

For individual LB (Luria Broth) agar plates: 

1. Pour sterile warm LB agar (about 25 ml) into a 

Petri dish dry opened and at room temperature 

under UV light for about 30 min. 

2. Add 40 μl of X-Gal Solution (20 mg/ml), ready-touse 

and 40 μl of 100 mM IPTG Solution, ready-touse 

(#R1171). 

3. Spread evenly on the plate with a sterile spatula. 

For batch use, add the following directly per 1 ml of 

the liquid LB agar (kept at about 50°C): 

1. 1 μl of X-Gal Solution, ready-to-use (20 mg/ml), 

(#R0941). 

2. 1μl of 100 mM IPTG Solution, ready-to-use (#R1171). 

3. Mix well and pour 25 ml of prepared LB agar into 

each Petri dish. 

4. Dry opened LB plates at room temperature under UV 

light for about 30 min. 

3.8. Analysis of recombinant clones 

Analyze 3-5 white colonies for the presence and 

orientation of the DNA insert using one of the following 

methods. 

Restriction analysis. Isolate plasmid DNA from 

an overnight bacterial culture using a convenient 

plasmid miniprep method such as the GeneJET 

Plasmid miniprep Kit (#K0502). Use FastDigest 

restriction enzymes (see p.2) to digest DNA from 

recombinant clones in just 5 min. 

Sequencing. Isolate plasmid DNA from an overnight 

bacterial culture using a reliable plasmid miniprep 

method such as GeneJET Plasmid Miniprep Kit 

(#K0502). Sequence the insert using appropriate 

sequencing primers. 

Colony PCR. Use the following protocol for colony 

screening by PCR. 

1. Prepare enough PCR master mix for the number of 

colonies analyzed plus one extra. For each 20 μl of 

reaction, mix the following reagents: 

Component 

Taq DNA 

Polymerase or 

Dreamtaq DNA 

Polymerase 

PCR Master 

Mix (2X) or 

Dreamtaq PCR 

Master Mix 

(2X) 

10X Taq Buffer 2 μl – 

dNTP Mix, 2mM each 2 μl – 

25 mM MgCl2 1.2 μl – 

M13/pUC Reverse Sequencing 

Primer (#SO101) 

0.6 μl (10 μM) 0.6 μl (10 μM) 

M13/pUC Forward Sequencing 


0.6 μl (10 μM) 0.6 μl (10 μM) 

Taq DNA Polymerase (#EP0402) 0.1 μl (0.5 u) – 

PCR Master Mix (2X) (#K0171) – 10 μl 

Water, nuclease-free (#R0581) to 20 μl to 20 μl 

Total volume 20 μl 20 μl 

2. Mix thoroughly, spin briefly and aliquot 20 μl of the 

mix into the PCR tubes on ice. 

3. Pick up an individual colony with a sterile pipette 

tip and resuspend it in 20 μl of the PCR master 

mix. Make a short streak with the same tip over a 

culture plate to save the clone for repropagation. 

4. Perform PCR: 95ºC, 3 min; 94ºC, 30 s; 56°C*, 30 

s; 72°C 1 min/kb; 30 cycles. 

5. Analyze on a gel for the presence of a PCR product 

of the expected length. 

* For recommended primer pair only. Annealing temperature 

might vary depending on primer pair used.


1 

Low yield or no 

transformants 

Assess ligation 

reaction 

efficiency 

Successful 

ligation 

1.1 

Transformation 

problem 

2.1 

Vector 

recircularization 

Assess ligase 

activity 

2 

Empty vector 

(no insert) 

Unsuccessful 

ligation 

2.2 

Incomplete 

cleavage of vector 

Inactive Ligase Active Ligase 

1.3.1 

Incorrect RE 

chosen for cloning 

1.3.3 

Inefficient 

blunting 

1.3.5 

Enzyme 

contamination 

Cloning problem 

3 

Incorrect 

construct 

1.2 

Low DNA 

quality 

1.3 

Incompatible 

DNA ends 

3.1 

Non-specific PCR 

product cloned 

3.2 

Enzyme 

contamination 

1.3.2 

Vector and insert 

are 

unphosphorylated 

1.3.4 

Inefficient 

cleavage of PCR 

product 

4 

Sequence errors 

in insert 

4.1 

Low fidelity 

PCR 

4.2 

UV damage to 

DNA fragment 

4.3 

Errors in PCR 

primer sequence 

5 

Colonies w/o 

plasmid 

5.1 

Low antibiotic 

concentration 

5.2 

Satellite 

colonies 


298 

Table 3.3. Troubleshooting guide for molecular cloning. 


1. Low yield or no 

transformants 


1.1. Inefficient transformation. 

1.1.1. Low transformation efficiency of competent E. coli cells. 

Check transformation efficiency with 0.1 ng of a supercoiled vector DNA; e.g., pUC19 (#SD0061). The competent 

cells should yield at least 1x10 6 transformants per μg of supercoiled DNA, which corresponds to 100 colonies, when 

0.1 ng of plasmid had been used for transformation. 

1.1.2. T4 DNA Ligase reduces transformation efficiency. 

Prior to transformation it is recommended to inactivate T4 DNA ligase by chloroform extraction or spin column 

purification. For commercial competent cells follow suppliers recommendation. 

1.1.3. Excessive amount of ligation mixture used for transformation. 

Do not use more than 5 μl of ligation mixture for 50 μl of chemically competent cells and 1 μl for electrocompetent cells. 

1.1.4. Cloned sequence is not tolerated by E.coli. 

Check the target sequence for strong E.coli promoters or other potentially toxic elements, as well as inverted 

repeats. In cases where the product of a cloned gene is toxic to the host, use promoters with a very low expression 

background or choose a low copy plasmid as cloning vehicle. 

1.1.5. Excessive amount of ligase used for ligation. 

Do not use more ligase than is recommended in the protocol (see p.294) relative to the vector and insert 

concentration. For easier manipulation, we provide T4 DNA Ligase at 1 u/μl (#EL0016). 

1.2. Low DNA quality. 

1.2.1. DNA contains reaction inhibitors (check according to p.294). 

Ensure DNA is free of contaminants (e.g., excess salts, EDTA, proteins, phenol, etc.) that may inhibit ligation. 

Gel purify and/or phenol/chloroform extract the vector and insert prior to ligation. 

1.2.2. DNA was damaged by UV light during excision from the agarose gel. 

Use a long wavelength UV (360 nm) light-box when excising DNA from the agarose gel. When using a shortwavelength 

(254-312 nm) light-box, limit DNA exposure to UV to a few seconds. Keep the gel on a glass or plastic 

plate during UV illumination. Alternatively, use dyes visible in ambient light to visualize DNA in standard 

agarose gels (1, 2). 

Another method to avoid exposure to UV is to load your sample in two or more lanes and then cut and stain only 

one lane with ethidium bromide after electrophoresis. Use this stained lane as a reference for excising the DNA from 

thunstained lane that is not exposed to UV light. 

1.3. Incompatible DNA ends. 

1.3.1. Incorrect restriction enzyme chosen for DNA digestion. 

Incompatible vector and insert ends. Recheck the cloning strategy and choose restriction enzymes generating 

compatible overhangs for ligation. 

1.3.2. Vector and insert are nonphosphorylated. 

If using dephosphorylated vectors, make sure the insert possesses phosphates. PCR products generally lack 

phosphate groups and need to be phosphorylated with T4 Polynucleotide Kinase (#EK0031) prior to ligation. The 

CloneJET PCR Cloning Kit (#K1231) is compatible with both phosphorylated and dephosphorylated DNA fragments. 

1.3.3. Inefficient blunting of DNA ends. 

Use the appropriate DNA blunting method for the DNA fragment ends. See p.295 for DNA blunting protocols and 

recommendations. 

1.3.4. Inefficient cleavage of PCR product. 

When introducing restriction enzyme sites into primers for subsequent digestion and cloning, refer to the Table 1.10. 

on p.169 to define the number of extra bases required for efficient cleavage. 

Prior to digestion remove the active thermophilic DNA polymerase from the PCR mixture. DNA polymerases may alter 

the ends of the cleaved DNA and reduce the ligation yield. 

After digestion, gel-purify the PCR product to remove short DNA fragments, which compete with the insert in the 

ligation reaction. 

1.3.5. Contaminated enzymes used for cloning. 

Use only the highest quality enzymes for cloning; e.g., LO-tested enzymes (see p.4) and exclude any possibility of 

endo-, exo-nuclease and phosphatase contamination in enzyme preparations. 

(continued on next page)

Table 3.3. Troubleshooting guide for molecular cloning. 


2. Empty vector 

(no insert) 

3. Incorrect constructs 

4. Sequence errors in 

insert 

5. Colonies without 

plasmid 

References 

1. Rand, K.N., Crystal Violet can be used to Visualize DNA 

Bands during Gel Electrophoresis and to Improve Cloning 

Efficiency, Elsevier Trends Journals Technical Tips Online, 

T40022, 1996. 

2. Adkins, S., Burmeister,M., Visualization of DNA in agarose 

gels and educational demonstrations, Anal. Biochem., 

240 (1), 17-23, 1996. 

2.1. Vector recircularization. 

Dephosphorylate the vector with FastAP Thermosensitive Alkaline Phosphatase (#EF0651) prior to ligation (see 

protocol on p.295). Vector dephosphorylation is recommended in all cases, including cloning strategies where the 

vector ends are incompatible for recircularization. Ensure the phosphatase is completely inactivated or removed after 

dephosphorylation. 

2.2. Incomplete cleavage of the vector. 

Check the cleavage efficiency on an agarose gel. If it is difficult to achieve complete cleavage, gel-purify the linear 

form of the vector using the GeneJET Gel Extraction Kit (#K0691). 

3.1. Non-specific PCR product cloned. 

Gel-analyze the PCR product prior to ligation. If non-specific PCR products or primer-dimers were generated during 

the PCR reaction, gel-purify the target PCR product. Smaller DNA fragments present in the PCR mixture are ligated 

more efficiently with the cloning vector and outcompete the target PCR products. 

Gel-purify the PCR product if the PCR template encodes ß-lactamase to avoid background colonies on LB-ampicillin 

agar. If the template and expected PCR product are of similar size, digest the template within the ampicillin 

resistance gene following the PCR reaction; e.g., with PdmI. 

3.2. Truncated insert due to contaminating endo- or exonucleases. 

Use only the highest quality enzymes for cloning; e.g., LO-tested enzymes (see p.4) and exclude any possibility of 

endo- and exonuclease contamination in enzyme preparations. 

4.1. Low fidelity DNA polymerase was used in PCR. 

If the PCR product will be used for cloning, it is always recommended to use a high fidelity DNA polymerase with 

proofreading activity such as Phusion High Fidelity DNA Polymerase. 

4.2. DNA was damaged by UV light during the excision from agarose gel. 

Use a long wavelength UV (360 nm) light-box when excising DNA from the agarose gel. When a short-wavelength 

(254-312 nm) light-box is used, limit DNA exposure to UV to a few seconds. Keep the gel on a glass or on plastic plate 

during UV illumination. Alternatively, use dyes visible in ambient light to visualize DNA in standard agarose gels (1, 2). 

4.3. Errors in PCR primers. 

If the cloned PCR product contains sequence errors or is missing 5’ bases and the same error persists in more than 

one clone, re-order the PCR primers from a reliable supplier and repeat the procedure starting from the PCR step. 

5.1. Insufficient amount of antibiotic in agar medium. 

Use 100 μg/ml of ampicillin in LB-ampicillin agar plates. Allow the LB medium to cool to 55°C before adding the 

antibiotic. Ampicillin is sensitive to light – long-term exposure to light can lead to low ampicillin concentration in 

plates. 

5.2. Satellite colonies. 

Some fast growing E.coli strains (e.g., C600) degrade ampicillin quickly, which leads to formation of smaller satellite 

colonies around transformants after >16 hours of incubation. Use shorter incubation times and do not use small 

satellite colonies for clone analysis. 

5.3. E.coli strain is resistant to the same antibiotic as the plasmid vector. 

Check genotype of E.coli strain used. For example XL-1 Blue is resistant to Tc, ER2267 is resistant to Km. 


300 


Thermo Scientific Nucleic Acid 

Purification Products 

Selection Guide ................................................................................................... 303 

Products .............................................................................................................. 304 

GeneJET Plasmid Miniprep Kit ....................................................................................304 

GeneJET Plasmid Midiprep Kit ....................................................................................305 

GeneJET Plasmid Maxiprep Kit ...................................................................................306 

GeneJET RNA Purification Kit ..................................................................................... 307 

GeneJET Whole Blood RNA Purification Mini Kit .......................................................308 

GeneJET Plant RNA Purification Mini Kit ...................................................................309 

GeneJET Genomic DNA Purification Kit ..................................................................... 310 

Genomic DNA Purification Kit ..................................................................................... 311 

GeneJET Whole Blood Genomic DNA Purification Mini Kit ....................................... 312 

GeneJET Plant Genomic DNA Purification Mini Kit.................................................... 313 

GeneJET Viral DNA and RNA Purification Kit ............................................................. 314 

Viral DNA/RNA Purification Kit (IVD) .......................................................................... 315 

GeneJET PCR Purification Kit ...................................................................................... 316 

GeneJET Gel Extraction Kit ......................................................................................... 317 

Silica Bead DNA Gel Extraction Kit ............................................................................. 318 

Agarase ......................................................................................................................... 319 

Related Products by Application ........................................................................ 320 

Protocols and Recommendations ...................................................................... 322 

4.1. Purification of genomic DNA from mouse tails with Proteinase K .................................322 

4.2. Purification of DNA from cultured eukaryotic cells with Proteinase K ...........................322 

4.3. Casting high quality agarose gels ..............................................................................322 

4.4. Recovery of DNA from LM agarose gels with Agarase ................................................322 

4.5. Nucleic acid precipitation from diluted solutions with Glycogen ................................... 323 

4.6. Removal of template DNA after in vitro transcription ................................................... 323 

4.7. Removal of genomic DNA from RNA preparations ....................................................... 323 

4.8. PCR product clean-up prior to sequencing ................................................................. 323 

4.9. Phenol/chloroform extraction and ethanol precipitation .............................................. 323 

Solutions for High Yield 

Nucleic Acid Purification: 

www.thermoscientific.com/nap 


302 

Reliable DNA and RNA Purification 

The Thermo Scientific GeneJET product line 

contains essential nucleic acid purification 

tools to support and facilitate your experimental 

workflow. With more than 30 years of 

experience in developing and manufacturing 

molecular biology products, we see a growing 

demand for simple and reliable tools for DNA 

and RNA sample preparation. The GeneJET 

DNA and RNA purification kits address the 

need for high-purity nucleic acids ready to use 

in downstream applications and offer detailed 

protocols for routine and difficult sample types. 

Miniprep 

Plasmid 

DNA 

Midiprep 


Maxiprep 

Cells & 

Tissues 

RNA 

Features 

Trusted silica membrane technology in 

the form of a convenient spin column for 

purification of DNA and RNA. 

Isolate nucleic acids with superior purity and 

yields compared to other kits on the market. 

Purified nucleic acids are ready to use in 

common downstream applications. 

GeneJET kits have been extensively tested on 

a wide variety of sample types and include 

specialized protocols. 

Total RNA 

Blood 

RNA 

Plant 

RNA 

Nucleic Acid 

Purification 

Cells & 

Tissues 

DNA 

Genomic 

DNA 

Blood 

DNA 

Plant 

DNA 

Viral DNA 

and RNA 

Serum & 

Plasma 

From 

PCR 

DNA 

Fragments 

From 

Gels


Applications Products to use Cat. # Page 

Isolation of plasmid DNA 

Isolation of RNA 

Isolation of genomic DNA 

Isolation of viral DNA and RNA 

PCR clean-up 

Extraction of DNA from 

agarose gel 

Removal of RNA from DNA preparations 

Removal of nucleotides 

Removal of primers 

Standard agarose gel 

Low melting point agarose gel 




GeneJET RNA Purification Kit K0731/2 307 

GeneJET Whole Blood RNA Purification Mini Kit K0761 308 

GeneJET Plant RNA Purification Mini Kit K0801/2 309 

GeneJET Genomic DNA Purification Kit K0721/2 310 

Genomic DNA Purification Kit K0512 311 

GeneJET Whole Blood Genomic DNA Purification Mini Kit K0781/2 312 

GeneJET Plant Genomic DNA Purification Mini Kit K0791/2 313 

Proteinase K (recombinant), PCR grade EO0491/2 270 

GeneJET Viral DNA and RNA Purification Kit K0821 314 

Viral DNA/RNA Purification Kit, (IVD)* DK0011 315 


FastAP Thermosensitive Alkaline Phosphatase EF0651/2/4 242 


Exonuclease I EN0581/2 258 




TopVision Agarose R0491/2 427 



Agarase EO0461 319 

TopVision Low Melting Point Agarose R0801 427 

RNase A, DNase and protease-free EN0531 262 

RNase T1 EN0541/2 263 

RNase A/T1 Mix EN0551 264 

RNase I EN0601/2 265 

Removal of DNA from RNA preparations DNase I, RNase-free EN0521/3/5 255 

Removal of proteins from DNA and RNA preparations Proteinase K (recombinant), PCR grade EO0491/2 270 

DNA and RNA precipitation 

* available in EU countries only 

Glycogen, RNA grade R0551 423 

Glycogen, molecular biology grade R0561 423 

3 M Sodium Acetate Solution, pH 5.2 R1181 421 


304 

Products 

GeneJET Plasmid Miniprep Kit 

#K0502 for 50 preps 



GeneJET Plasmid Midiprep Kit p.305 

GeneJET Plasmid Maxiprep Kit p.306 








Resuspend Cells 


Lyse and Neutralize 

Bind 

Wash x 2 

Elute 

Overview of purification procedure. 

M 1 M 2 C 1 2 3 4 5 6 M1 M2 

Fast clone analysis. 

A 2.3 kb PCR fragment was cloned into the pUC19 vector. Plasmid 

DNA from overnight bacterial cultures of recombinant clones was 

purified using the GeneJET Plasmid Miniprep Kit (#K0502) and 

analyzed by double digestion with FastDigest EcoRI and FastDigest 

HindIII in 5 min at 37°C. 

M 1, M 2 – ZipRuler Express DNA Ladder Set (#SM1373) 

C – control pUC19 DNA digested with FastDigest EcoRI and HindIII 

1-6 – Miniprep DNA from recombinant clones, double 

digested with FastDigest EcoRI and HindIII 

Description 

The Thermo Scientific GeneJET Plasmid Miniprep 

Kit is a simple, rapid and cost-effective system 

for the isolation of high quality plasmid DNA 

from recombinant E.coli cultures. The kit utilizes 

an exclusive silica-based membrane technology 

in the form of a convenient spin column. Each 

prep recovers up to 20 μg of high copy plasmid 

DNA. The purified DNA is ready for immediate 

use in all molecular biology procedures such 

as digestion with FastDigest and conventional 

restriction enzymes, PCR, in vitro transcription, 

transformation, and automated sequencing. 

Features 

Efficient – high yields of up to 20 μg of high 

quality plasmid DNA. 

Fast – procedure takes less than 14 min. 

Convenient – no phenol/chloroform 

extraction or alcohol precipitation required. 

Pure – purified DNA is immediately 

ready-to-use. 

Applications 

Fast isolation of high purity plasmid DNA 

suitable for all conventional molecular biology 

procedures, including: 

– FastDigest or conventional restriction 

digestion 

– automated fluorescent and radioactive 

sequencing 

– PCR 

– in vitro transcription 

– transformation 

– transfection of robust cell lines 


 

 

 

 

 

 

 

 

 

Electropherogram demonstrates the high quality sequencing 

data of plasmid DNA purified with the Thermo Scientific GeneJET 

Plasmid Miniprep Kit. 

Plasmid DNA was purified from E.coli using the GeneJET Plasmid Miniprep Kit and 

sequenced with a T7 sequencing primer. The sequencing reaction was analyzed on 

an ABI 3730 DNA Analyzer. More than 750 nucleotides can be read.

GeneJET Plasmid Midiprep Kit 




GeneJET Plasmid Maxiprep Kit p.306 









DNA, μg 

300 

250 

200 

150 

100 

50 

0 



Clear lysate by centrifugation 

Bind 

Wash x 3 

Elute 



Scientific 

vendor 

A 

vendor 

B 

High yields of plasmid purified with the Thermo Scientific GeneJET Plasmid 

Midiprep Kit. Yields of high-copy plasmid DNA obtained using the GeneJET Plasmid 

Midiprep Kit and kits from other vendors for mid-scale plasmid DNA purification at 

maximum recommended bacterial culture volumes. 

Description 

The Thermo Scientific GeneJET Plasmid Midiprep 

Kit is designed for medium scale isolation of 

high quality plasmid DNA from recombinant 

E.coli cultures. The kit utilizes silica-based 

membrane technology in the form of a 

convenient spin column. Each prep recovers 

up to 200 μg of high copy number plasmid 

DNA. The purified plasmid DNA can be used in 

a wide variety of molecular biology procedures 

such as restriction digestion, PCR, cloning, 

transformation, automated sequencing, in vitro 

transcription and transfection of robust cell 

lines. 

Features 

Efficient – high yields up to 200 μg of high 

quality plasmid DNA from 50 ml of bacterial 

culture. 

High concentration of DNA purified – 

>400 ng/μl. 

Convenient – spin column format, centrifuge 

and vacuum protocols. 

Fast – procedure takes 60 min, considerably 

faster than using gravity flow columns. 

vendor 

C 

vendor 

D 

DNA, ng/μl 

900 

800 

700 

600 

500 

400 

300 

200 

100 

0 



Applications 

Isolated plasmid DNA is ideal for all 

conventional molecular biology procedures, 

including: 

– FastDigest restriction enzyme digestion 

– automated fluorescent sequencing 

– PCR 

– cloning 





 

 

 

 

 

 

 

 

 

pre-assembled with collection tubes (15 ml) 

 

 

vendor 

A 

vendor 

B 

vendor 

C 

1st elution 

2nd elution 

vendor 

D 

High concentration of plasmid purified with the Thermo Scientific GeneJET 

Plasmid Midiprep Kit. Concentration of eluted DNA obtained after purification of 

high-copy plasmid DNA using different kits for mid-scale plasmid DNA purification at 

maximum recommended bacterial cell culture volumes. 


306 

GeneJET Plasmid Maxiprep Kit 




GeneJET Plasmid Midiprep Kit p.305 









Transfection efficiency, % 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 


cos-7 HeLa NIH 3T3 HEK 293 CHO 

Description 

The Thermo Scientific GeneJET Plasmid Maxiprep 

Kit is designed for large scale isolation of high 

quality plasmid DNA from recombinant E.coli 

cultures. The kit utilizes silica-based membrane 

technology in the form of a convenient spin 

column. Each prep recovers up to 750 μg of 

high copy plasmid DNA that can be used in a 

wide variety of molecular biology procedures 

such as restriction digestion, PCR, cloning, 

transformation, automated sequencing, in vitro 

transcription and transfection of robust cell lines. 

Features 

Efficient – high yields up to 750 μg of high 

quality plasmid DNA from 250 ml of bacterial 

culture. 

High concentration of DNA purified – 

>400 ng/μl. 

Convenient – spin column format, centrifuge 

and vacuum protocols. 

Fast – procedure takes 60 min, considerably 

faster than using gravity flow columns. 

Applications 

Isolated plasmid DNA is ideal for all 

conventional molecular biology procedures, 

including: 


– FastDigest restriction enzyme digestion 

– automated fluorescent sequencing 

– PCR 

– cloning 



CHR 

GeneJET 

Efficient transfection of robust cell lines with plasmid DNA purified using the Thermo 

Scientific GeneJET Plasmid Maxiprep Kit. Plasmid DNA, purified from E.coli cells using the 

GeneJET Plasmid Maxiprep Kit, gives transfection efficiencies comparable to chromatography 

purified, endotoxin-free plasmid DNA. 1 μg of the high-copy plasmid was transfected into 

various cell lines using the TurboFect Transfection Reagent (R0531). 

CHR – chromatography purified, endotoxin-free plasmid, GeneJET – plasmid purified using the 

GeneJET Plasmid Maxiprep Kit. 

DNA, μg 

800 

700 

600 

500 

400 

300 

200 

100 

0 




 

 

 

 

 

 

 

 

 

pre-assembled with collection tubes (50 ml) 

 

 

vendor 

A 



Clear lysate by centrifugation 

Bind 

Wash x 3 

Elute 


vendor 

B 

vendor 

C 

vendor 

D 

High yields of plasmid purified DNA with the Thermo Scientific 

GeneJET Plasmid Maxiprep Kit. 

Plasmid DNA was purified from E.coli cells using the GeneJET Plasmid 

Maxiprep Kit and kits of various vendors using recommended culture volumes 

and purification protocols.

GeneJET RNA Purification Kit 






RiboRuler Low Range RNA Ladder, 

ready-to-use p.386 

RiboRuler High Range RNA Ladder, 




Collect sample 

Lyse 

Bind 

Wash x 3 

Elute 


References 

1. Chomczynski, P. and Sacchi. N. Single-step method of 

RNA isolation by acid guanidinium thiocyanate-phenolchloroform 

extraction. Anal. Biochem. 162:156-159,1987. 

2. Boom, R., Sol C.J.A., Salimans, M.M.M. Jansen, C.L. 

Dillen, P.M.E.W. and van der Noordaa J. Rapid and simple 

method for purification of nucleic acids. J. Clin. Microbiol. 

28:495-503, 1990. 

Description 

The Thermo Scientific GeneJET RNA Purification 

Kit is a simple and efficient system for 

purification of RNA from a wide variety of 

samples including mammalian cell culture and 

tissues, blood, bacteria, and yeast. 

The kit utilizes a proprietary silica-based 


convenient spin column, eliminating the need 

for tedious cesium chloride gradients, alcohol 

precipitation or toxic phenol-chloroform 

extractions. 

All RNA molecules longer than 200 nucleotides 

can be isolated with the GeneJET RNA 

Purification Kit in less than 15 min. The purified 

high quality RNA can be used in a wide range 

of downstream applications such as RT-PCR, 

RT-qPCR, Northern blotting and other RNA-based 

analysis. 

Features 

Universal – can be used for both cell and 

tissue samples from a wide range of sources. 

Fast – procedure takes less than 15 min. 

Yields high quality total RNA ideal for most 


M 1 2 3 4 5 6 

Total RNA from various sources purified using 

the Thermo Scientific GeneJET RNA 

Purification Kit. 

1 μg of isolated total RNA was fractionated by agarose 

gel (1%) electrophoresis. 

M – RiboRuler High Range RNA Ladder (#SM1821) 

1 – mouse liver 

2 – mouse spleen 

3 – HeLa cells 

4 – Cos-7 cells 

5 – Saccharomyces cerevisiae 

6 – E.coli DH5cells 

Applications 

Fast and efficient purification of high quality 

RNA from: 

– whole blood 

– mammalian cell cultures 

– mammalian tissues 

– yeast 

– bacteria 


 

Wash Buffer 1 (concentrated) 

Wash Buffer 2 (concentrated) 

Proteinase K 

 

 

assembled with collection tubes 

 

 

[FU] 

300 

200 

100 

0 

RIN:10 

25 200 1000 4000 

Lenght, nt 

High integrity of total RNA isolated from HeLa 

cells using the GeneJET RNA Purification kit. 

RNA was analyzed on an A 

rRNA ratio [28S/18S] = 2/1 

RIN – RNA integrity number 


308 

GeneJET Whole Blood RNA Purification Mini Kit 












Collect cells 

Lyse 

Bind 

Wash x 3 

Elute 


Description 

The Thermo Scientific GeneJET Whole Blood 

RNA Purification Mini Kit is designed for rapid 

and efficient purification of high quality total 

RNA from whole blood and related body fluids. 




for expensive resins, toxic phenol-chloroform 

extractions, or time-consuming alcohol 

precipitation. 

The standard procedure takes less than 15 min. 

The purified high quality RNA can be used in a 

wide range of downstream applications such as 

RT-PCR, RT-qPCR, Northern blotting and other 

RNA-based analysis. 

Features 

Efficient – purification of up to 5 μg total RNA 

from 500 μl of human blood. 

Convenient – spin columns are capped and 

assembled with collection tubes. 

Pure – isolated RNA is free of enzyme 

inhibitors and proteins, is ready to use in all 

demanding applications, and is validated in 

cDNA synthesis and RT-qPCR. 

Fast and simple – procedure takes less 

than 15 min and does not involve organic 

extraction or differential RBC lysis. 

Compatible – ideal for a wide variety of 

samples including human and animal blood 

treated with EDTA, citrate or heparin; buffy 

coat; bone marrow, and related body fluids. 

RIN 8.4 8.3 9.7 8.3 8.3 8.0 8.9 8.6 8.1 8.0 8.3 8.2 

fresh blood 24 h at 4°C 7 d at -20°C 

M 1 2 3 4 5 6 7 8 9 10 11 12 

Stabilization of blood samples by Lysis Buffer. 

RNA purified from blood samples was stored at various 

temperatures in Lysis Buffer. 500 μl of whole blood 

was centrifuged, cells were resuspended in 600 μl of 

Lysis Buffer and either used immediately (lanes 1-4) 

or stored for 24 h at 4°C (lanes 5-8), or for 7 days 

at -20°C (lanes 9-12) before processing with the 

GeneJET Whole Blood RNA Purification Mini Kit. RNA 

integrity was analyzed using Agilent 2100 Bioanalyzer. 

Applications 

Fast extraction of high purity RNA suitable 

for all conventional molecular biology 


– RT-PCR 

– RT-qPCR 

– Northern blotting 

– nuclease protection assay 


 

 

 

 

 

(pre-assembled with collection tubes) 

 

 

 

M 1 2 3 4 5 

Total RNA purified from human blood using 

the Thermo Scientific GeneJET Whole Blood 

RNA Purification Mini Kit and kits from other 

vendors. 

5 μl (10%) of RNA isolated from 500 μl of human blood 

was fractionated by agarose gel (1 %) electrophoresis. 

1 – Thermo Scientific 

2-4 – Various vendors 

Reproducible RNA yields and purity. 

RNA purified from 6 human blood samples using the GeneJET 

Whole Blood RNA Purification Mini Kit. 1.5 μl of isolated RNA 

was analyzed on NanoDrop spectrophotometer.

GeneJET Plant RNA Purification Mini Kit 












Prepare sample 

Lyse 

Bind 

Wash x 3 

Elute 


Description 

The Thermo Scientific GeneJET Plant RNA 

Purification Mini Kit is designed for rapid and 

efficient purification of high quality total RNA 

from a wide variety of plant species and 

tissue types. 







The standard procedure takes less than 20 min 

following cell lysis. Purified high quality RNA 

can be used in a wide range of downstream 

applications such as RT-PCR, RT-qPCR, 

Northern blotting and other RNA-based analysis. 

RNA yields vary between different species, 

tissues and age of tissue sample. 

Features 

Fast – procedure completed in 20 min 

following cell lysis. 

Convenient – protocol does not include 

clogging-prone lysate filtration step. 

High yields – up to 65 μg of high purity and 

high integrity RNA from 50 mg plant sample. 

Compatible with a wide variety of samples 

(leaves, roots, sprouts and other 

plant tissues). 

RNA purified from tomato leaves using 

different kits. 

1 μl (2% of eluate) of RNA isolated using the GeneJET 

Plant RNA Purification Mini Kit and kits from other 

vendors were fractionated on a 1% agarose gel. 

M – RiboRuler High Range RNA Ladder (#SM1821) 

1 – Thermo Scientific 

2-5 – Various vendors 

Applications 

Fast extraction of high purity RNA suitable 

for all conventional molecular biology 


– RT-PCR 

– RT-qPCR 

– Northern blotting 

– nuclease protection assay 


 

 

 

 

 

pre-assembled with collection tubes 

 

 

 

High integrity RNA isolated from difficult to 

process samples using the Thermo Scientific 

GeneJET Plant RNA Purification Mini Kit. 

1 μl of total RNA isolated from 50 mg of various 

plants and plant tissues was analyzed with the Agilent 

2100 Bioanalyzer. 

1 – Corn roots, 2 – Canola roots, 3 – Sunflower stalk, 

4 – Canola stalk, 5 – Cucumber fruit, 6 – Tomato 

fruit, 7 – Sunflower seeds, 8 – Canola seeds, 

9 – Corn seeds 


310 

GeneJET Genomic DNA Purification Kit 




GeneRuler 1 kb Plus DNA Ladder, 






Lyse 

Bind 

Wash x 2 

Elute 


Description 

The Thermo Scientific GeneJET Genomic 

DNA Purification Kit is designed for rapid and 

efficient purification of high quality genomic 

DNA from whole blood, various mammalian 

tissues and cell culture samples and bacteria. 







The standard procedure takes less than 20 min 

after cell lysis. The purified genomic DNA is up 

to 50 kb in size with dominating fragments of 

20-30 kb. Isolated DNA can be used directly for 

PCR, Southern blotting and enzymatic reactions. 

M 1 2 3 4 5 6 7 8 M 

Genomic DNA from various sources purified 

using the Thermo Scientific GeneJET Genomic 

DNA Purification Kit. 

150 ng of isolated genomic DNA from each source was 

fractionated by agarose gel (0.5%) electrophoresis. 

M – Lambda DNA/HindIII Marker (#SM0101) 

1 – human bone marrow 

2 – human blood 

3 – rat spleen 

4 – mouse heart 

5 – mouse liver 

6 – HeLa cells 

7 – Jurkat cells 

8 – E.coli DH5cells 

Features 

Universal – can be used for both cell and 

tissue samples from a wide range of sources. 

Fast – procedure completed 20 min after 

lysis step. 

Efficient – yields high quality genomic 

DNA of high molecular weight and minimal 

degradation suitable for most downstream 

applications. 

Applications 

Fast isolation of genomic DNA from: 

– whole blood 

– mammalian cell cultures 


– bacteria 


 

 

Digestion Solution 

 

Wash Buffer I (concentrated) 

Wash Buffer II (concentrated) 

Elution Buffer 

GeneJET Genomic DNA Purification Columns 


 

 

Reference 

1. Boom, R., Sol C.J.A., Salimans M.M.M., Jansen C.L., 

P.M.E.W. Dillen, and van der Noordaa J. Rapid and simple 

method for purification of nucleic acids. J. Clin. Microbiol. 

28:495-503, 1990.

Genomic DNA Purification Kit 


one preparation is sufficient for: 

200 μl of whole blood, 

25-30 mg of mammalian tissue, 

50-100 mg of plant tissue, 

0.4-0.6x10 6 of cultured cells or 

10-20 mg of bacteria. 


RNase A, DNase and protease-free p.262 


Glycogen, molecular biology grade p.423 


Add Lysis Solution to blood 


Add chloroform 

Invert 3-5 times 


Transfer the upper phase to a 

new tube 

Add Precipitation Solution 

mix gently 


Discard supernatant 

Dissolve in NaCl Solution 

Precipitate with 96% ethanol 



Wash pellet with 70% cold ethanol 

Disolve in water or TE 


DNA purification from whole blood. 

Description 

The Thermo Scientific Genomic DNA Purification 

Kit is a simple and rapid system for high 

quality genomic DNA purification from various 

sources including whole blood, serum, cell lines, 

bacterial cells, plant and mammalian tissues. 

The kit is based on selective detergentmediated 

DNA precipitation from crude 

lysate. The entire procedure is rapid – only 

20-25 min – with a typical yield of 2-10 μg 

genomic DNA from 0.2 ml of blood. High 

molecular weight genomic DNA purified with 

the kit is ideal for direct use in all common 

molecular biology applications: PCR, FastDigest 

and conventional restriction digestion, cloning, 

DNA sequencing, and Southern blot analysis. 

Features 

Efficient – 2-10 μg of genomic DNA from 

0.2 ml of blood. 

Fast – procedure takes approximately 

20 min. 

Universal – purifies genomic DNA from 

various sources. 

Flexible – easy to scale up and down. 

Safe – does not involve phenol extraction. 

Applications 

Purification of high-quality genomic DNA from: 

– whole blood or serum 

– cell culture 

– plant tissues 


– epithelium samples 

– bacteria 


Lysis Solution 

Precipitation Solution 

NaCl Solution 

 

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M 

Genomic DNA from various sources purified with the Thermo Scientific Genomic 

DNA Purification Kit. 

Agarose gel (0.7%) electrophoresis analysis of undigested (odd lane numbers) and EcoRI 

(#ER0271) digested (even lane numbers) DNA samples. 


1, 2 – human blood 

3, 4 – human lymphocytes 

5, 6 – human placenta 

7, 8 – mouse liver 

9, 10 – Hedera helix 

11, 12 – Haemophilus influenzae 

13, 14 – E.coli ER2267 

15, 16 – Bacillus subtilis 


312 

GeneJET Whole Blood Genomic DNA Purification Mini Kit 










Lyse 

Bind 

Wash x 2 

Elute 


Description 

The Thermo Scientific GeneJET Whole Blood 

Genomic DNA Purification Mini Kit is designed 

for rapid and efficient purification of high quality 

genomic DNA from whole blood and related 

body fluids. 







The standard procedure takes less than 20 

min following cell lysis and yields purified DNA 

greater than 30 kb in size. Isolated DNA can 

be used directly in PCR, Southern blotting and 

enzymatic reactions. 

Features 

Efficient – high yields of high molecular 

weight genomic DNA. 

Fast – 20 min procedure following cell lysis. 



Pure – isolated DNA is free of enzyme 

inhibitors, RNA and proteins, and is ready 

to use in common downstream applications 

such as qPCR, endpoint PCR, multiplex PCR, 

and restriction enzyme digests. 

Compatible – ideal for wide variety 

of samples including fresh or frozen 

anticoagulated mammalian blood (treated 

with EDTA, citrate or heparine), aged blood, 

buffy coat, bone marrow, body fluids. 

Mean Yield (pg) 

Accurate evaluation of genomic DNA yields. 

Genomic DNA was purified from human blood using the GeneJET 

Whole Blood Genomic DNA Purification Mini Kit and kits from other 

vendors. DNA concentration was estimated using a NanoDrop 

spectrophotometer (Thermo Scientific). 200 pg was used as 

a template in qPCR with primers against a single copy gene. 

Matching yields obtained by qPCR-based absolute quantification 

and spectrophotometrical evaluation indicate the absence of 

reaction inhibitors and contaminating RNA. 

Applications 

Isolated genomic DNA is ideal for use in 

common molecular biology procedures, 

including: 

– PCR and qPCR 

– FastDigest and conventional restriction 

digestion 

– Southern blotting 


 

 

 

 

 

 


 

 

M 1 2 3 4 M 

Genomic DNA purified from various sources 

using the Thermo Scientific GeneJET Whole 

Blood Genomic DNA Purification Mini Kit. 

200 ng of isolated genomic DNA from each 

source was fractionated by agarose gel (0.5%) 



1 – Human whole blood 

2 – Human buffy coat 

3 – Human bone marrow 

4 – Avian whole blood

GeneJET Plant Genomic DNA Purification Mini Kit 








Prepare sample 

Lyse 

Precipitate 

Bind 

Wash x 2 

Elute 


Description 

The Thermo Scientific GeneJET Plant Genomic 

DNA Purification Mini Kit is designed for rapid 

and efficient purification of high quality genomic 

DNA from a wide variety of plant species and 

tissue types. 





extractions or time-consuming alcohol 

precipitation. The standard procedure takes 

less than 30 min following cell lysis and yields 

purified DNA greater than 30 kb in size. DNA 

yields vary between different species and 

tissues depending on genome size, ploidy, cell 

number and the age of the tissue sample. The 

typical yield from a standard tissue source (e.g., 

young wheat leaves) is 30-32 μg from 

100 mg of tissue. Isolated DNA can be used 

directly for PCR, qPCR, Southern blotting and 

enzymatic reactions. 

Features 

Fast – procedure completed in 30 min 

following cell lysis. 

Convenient – protocol without clogging-prone 

lysate filtration step. 

High yield – up to 32 μg of high quality DNA 

from 100 mg sample, including nuclear, 

mitochondrial and chloroplast DNA. 

Compatible with a wide variety of sample 

types (leaves, roots, sprouts and other parts 

of various plants). 

Applications 

Isolated genomic DNA is ideal for use in 

common molecular biology procedures, 

including: 

– PCR and qPCR 

– FastDigest and conventional restriction 

digestion 

– Southern blotting 


 

 

 

 

 

 

 

 

 


 

 

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M 

Genomic DNA purified from various sources using the Thermo Scientific GeneJET Plant Genomic 

DNA Purification Mini Kit. 

200 ng of isolated genomic DNA from each source was fractionated by agarose gel (0.5%) electrophoresis. 

M – Lambda DNA/HindIII Marker (#SM0101), 1 – Arabidopsis, 2 – Tobacco leaves, 3 – Tomato leaves, 

4 – Spinach leaves, 5 – Wheat leaves, 6 – Canola leaves, 7 – Onion leaves, 8 – Rice leaves, 9 – Corn seeds, 

10 – Sunflower stalk, 11 – Corn roots, 12 – Beet roots, 13 – Cucumber fruit, 14 – Sugar cane. 


314 

GeneJET Viral DNA and RNA Purification Kit 



Viral DNA/RNA Purification Kit (IVD) p.315 



Lyse 

Bind 

Wash x 3 

Elute 


Description 

The Thermo Scientific GeneJET Viral DNA and 

RNA Purification Kit is designed for rapid and 

efficient purification of high quality viral nucleic 

acids from a variety of human and mammalian 

fluid samples such as plasma, serum, whole 

blood, saliva, nasal and buccal swabs, 

urine, urogenital swabs, and milk. The kit is 

compatible with common sample preservatives 

such as EDTA, citrate and heparin. 

Purified viral nucleic acids are free of proteins, 

nucleases and other contaminants or inhibitors 

of downstream applications. Isolated DNA and 

RNA can be directly used in PCR, qPCR or other 

nucleic acid based assays. 

Principle 



convenient spin column. Viral nucleic acids from 

lysed samples bind to the column membrane 

while impurities are effectively removed during 

subsequent washing and centrifugation steps. 

Ready-to-use nucleic acids then are eluted from 

the column. 

Features 

Efficient purification of viral DNA and RNA 

from human and mammalian plasma, serum, 

blood, and cell-free samples. 

Linear yields from a wide range of viral titers. 

Tested with a variety of DNA and RNA viruses 

(HIV, HBV, HCV, HPV, CMV, EBV). 

Downstream compatibility with (RT)-PCR, 

(RT)-qPCR and other nucleic acid based 

assays. 

Log 10 copies/ml 

8 

7 

6 

5 

4 

3 

2 

1 

0 

HBV HCV CMV EBV 


vendor A 

vendor B 

vendor C 

Highly efficient purification of viral nucleic 

acids from various sample types. 

Different amounts of HBV, HCV, CMV and EBV infected 

human plasma or blood samples were purified using the 

GeneJET Viral DNA and RNA Purification kit or kits from 

other suppliers. Isolated viral nucleic acids were quantified 

using a real-time PCR detection assay. 

Applications 

Downstream use in qualitative and 

quantitative nucleic acid based assays, e.g., 

(RT)-PCR, (RT)-qPCR. 


 

 

 

 

 

 

 

 

Tubes 

 

 

Result HCV, log IU/ml 

A 

Result HBV, log IU/ml 

B 

8 

7 

6 

5 

4 

3 

2 

1 

0 

R 

0 1 2 3 4 5 6 7 8 

2 = 0.9983 

Input HCV, log IU/ml 

8 

7 

6 

5 

4 

3 

2 

1 

0 

R 

0 1 2 3 4 5 6 7 8 

2 = 0.9985 

Input HBV, log IU/ml 

Linear performance across a wide range of viral 

nucleic acid concentrations. 

Real-time PCR detection of Viral RNA (A) and DNA (B) 

purified with the GeneJET Viral DNA and RNA Purification 

Kit. Samples of various HBV and HCV titers were produced 

by diluting infected plasma of known viral titer with noninfected 

plasma. 

A: Detection of RNA from various HCV sample titers. 

B: Detection of DNA from various HBV sample titers. 

Reference 

1. Boom, R., C.J.A. Sol, M.M.M. Salimans, C.L. Jansen, P.M.E.W. 

Dillen, and J. van der Noordaa. Rapid and simple method 

for purification of nucleic acids. J. Clin. Microbiol. Hazarddetermining 

component of labeling: Guanidinium chloride 

28:495-503, 1990.

Viral DNA/RNA Purification Kit (IVD)* 

*Available in EU countries only. 

#DK0011 for 50 preps 


GeneJET Viral DNA and RNA Purification Kit p.314 


Lyse 

Bind 

Wash x 3 

Elute 


Description 

The Thermo Scientific Viral DNA/RNA 

Purification Kit is a simple and easy-to-use 

system for efficient purification of viral 

nucleic acids from human plasma or serum 

samples for in vitro diagnostic purposes. 

The purification system utilizes a proprietary 

silica-based membrane technology in the 

form of a convenient spin column. The entire 

purification procedure takes approximately 

30 min to process 1-4 samples. Purified viral 

DNA and RNA are compatible with downstream 

qualitative and quantitative in vitro diagnostic 

workflows such as PCR, qPCR, RT-PCR and 

RT-qPCR. The kit is compatible with citrateor 

EDTA-treated human plasma and serum 

samples. To avoid sample contamination and to 

ensure safe waste handling, fresh wash tubes 

are supplied for each wash step. 

Principle 

During the purification procedure, viral nucleic 

acids from lysed plasma or serum samples 

bind to the column membrane while impurities 

are effectively removed during subsequent 

wash steps. Ready-to-use nucleic acids are 

then eluted from the column. The purified viral 

nucleic acids are free of proteins, nucleases, 

and other contaminants or inhibitors of 


Result HCV, log IU/ml 

A 

Fluorescence (norm) 

A 

8 

7 

6 

5 

4 

3 

2 

1 

0 

R 

0 1 2 3 4 5 6 7 8 

2 = 0.9983 

0.40 

0.35 

0.30 

0.25 

0.20 

0.15 

0.10 

0.05 

0.00 

5 10 

Input HCV, log IU/ml 

15 20 25 30 35 40 45 

Cycle number 

Features 

Efficient purification of viral DNA and RNA 

from a wide range of virus concentrations. 

Compatible with fresh or frozen serum and 

plasma preserved with EDTA or citrate. 

Minimized risk of cross-contamination. 

CE-marked for compliance with EU Directive 

98/79/EC. 

Applications 

Isolation of viral nucleic acids from 200 μl 

of fresh or frozen human plasma and 

serum samples preserved with either 

EDTA or citrate. 

Downstream use in in vitro diagnostic 

qualitative and quantitative nucleic acid 

based assays (e.g., PCR, RT-PCR). 


 

 

 

 

 

 

 

 

 

Tubes 

 

 

 

Result HBV, log IU/ml 

8 

7 

6 

5 

4 

3 

2 

1 

0 

R 

0 1 2 3 4 5 6 7 8 

2 = 0.9985 

Input HBV, log IU/ml 

Linear performance across a wide range of viral nucleic acid concentrations. 

Real-time PCR detection of Viral RNA (A) and DNA (B) purified with the Viral DNA/RNA Purification Kit. Samples 

of various HBV and HCV titers were produced by diluting infected plasma of known viral titer with non-infected 

plasma. A: Detection of RNA from various HCV sample titers. B: Detection of DNA from various HBV sample titers. 

B 

Fluorescense (norm) 

B 

0.40 

0.35 

0.30 

0.25 

0.20 

0.15 

0.10 

0.05 

0.00 

5 10 

15 20 25 30 35 40 45 

Cycle number 


316 

GeneJET PCR Purification Kit 




TopVision Agarose p.427 

TopVision Low Melting Point Agarose p.427 

50X TAE Buffer p.371 

10X TBE Buffer p.371 

0.5 M EDTA, pH 8.0 p.421 

DNA Ladders & Markers pp.364-370 






Load PCR product 

Prepare for binding 

Bind 

Wash 

Elute 


Description 

The Thermo Scientific GeneJET PCR Purification 

Kit is designed for rapid and efficient purification 

of DNA from PCR and other enzymatic 

reaction mixtures. 



convenient spin column, eliminating the 

need for tedious resin manipulations or toxic 

phenol-chloroform extractions. The GeneJET 

PCR Purification Kit effectively removes primers, 

dNTPs, unincorporated labeled nucleotides, 

enzymes and salts from PCR and other reaction 

mixtures. The kit can be used for purification 

of DNA fragments from 25 bp to 20 kb with 

recovery rates up to 100%. Each GeneJET 

purification column has a total binding capacity 

of up to 25 μg of DNA and the entire procedure 

takes just 5 min. The purified DNA can be used 

in common downstream applications such 

as sequencing, restriction digestion, labeling, 

ligation, cloning, in vitro transcription, blotting or 

in situ hybridization. 

Label 

ROX-NHS 

Recovery rate, % 

100 

80 

60 

40 

20 

0 

before after 

Efficient removal of 

unincorporated label. 

100 bp 

500 bp 

400 bp 

1 kb 

Size 

Features 

Highly efficient – 90-100% recoveries in the 

range of 100 bp-10 kb. 



Fast – procedure takes 5 min. 

Pure DNA – A 260/A 280 > 1.8. 

Applications 

Fast and efficient purification of DNA fragments 

ideal for use in all conventional molecular 

biology procedures including: 


digestion 

– automated fluorescent or radioactive 

sequencing 

– PCR 


– in situ hybridization 

– labeling 

– cloning 


 

 

 

 

(preassembled with collection tubes) 

 

5.5 kb 10 kb 


vendor A 

vendor B 

Superior purification of large and small DNA fragments from PCR reaction mixtures 

using the Thermo Scientific GeneJET PCR Purification Kit.

GeneJET Gel Extraction Kit 








DNA Ladders & Markers pp.364-370 





Excise gel slice with DNA 

fragment 

Lyse 

Bind 

Wash 

Elute 


Description 

The Thermo Scientific GeneJET Gel Extraction 

Kit is designed for rapid and efficient 

purification of DNA fragments from standard or 

low-melting point agarose gels run in either TAE 

or TBE buffer. 



convenient spin column. The kit can be used 

to purify DNA fragments from 25 bp to 20 kb 

in size with recovery rates up to 95%. Each 

GeneJET purification column has a binding 

capacity of up to 25 μg of DNA and can process 

up to 1 g of agarose gel. The entire procedure 

takes just 15 min and the isolated DNA is ready 

to use in all common downstream applications 

including ligation, restriction digestion, PCR, 

sequencing and labeling. 

Features 

Highly efficient – 80-95% recoveries in the 

range of 100 bp – 10 kb. 



Fast – procedure takes 15 min. 

Pure DNA – A260/A280 > 1.8. 

Recovery rate, % 

100 

80 

60 

40 

20 

0 

100 bp 500 bp 1 kb 

Size 

5.5 kb 10 kb 

Superior extraction of large and small DNA fragments from agarose gels. 

Applications 

Fast and efficient extraction of high purity DNA 

fragments ideal for use in all conventional 

molecular biology procedures including: 


digestion 

– automated fluorescent or radioactive 

sequencing 

– PCR 

– in situ hybridization 

– labeling 

– cloning 


 

 

 

 

with collection tubes) 

 

Note 

 

with UV light when the gel-purified DNA 

fragment will be used in downstream 

cloning reactions. Use a long wavelength 

(360 nm) UV light-box during excision 

of the gel slice, minimize the UV exposure to 

a few seconds or keep the gel on a glass or 

plastic plate. 


vendor A 

vendor B 


318 

Silica Bead DNA Gel Extraction Kit 


(up to 5 μg of DNA per prep) 








T4 DNA Ligase 


p.239 



Excise gel slice with DNA 

fragment 

Add Binding Solution to the gel slice 


Add Silica Suspension 


Centrifuge 


Resuspend in wash buffer 

Centrifuge 

Discard wash buffer 

Repeat 3X 

Resuspend in TE buffer 


Centrifuge 

Transfer purified DNA to a 

fresh tube 



Description 

The Thermo Scientific Silica Bead DNA Gel 

Extraction Kit is a simple and efficient system for 

DNA extraction from agarose gels and reaction 

mixtures. The kit utilizes the modified protocol 

of Vogelstein and Gillespie (1). The extraction 

is based on the solubilization of the agarose 

gel and selective adsorption of nucleic acids 

on specially prepared silica particles at high 

salt concentration. Silica particles bound with 

DNA are washed to remove contaminants and 

pure DNA is eluted with TE buffer or water. This 

method requires few manipulations, and is both 

faster and easier to perform than organic-based 

extraction methods. Compared to column based 

gel-extraction kits, the Silica Bead DNA Gel 

Extraction Kit offers flexibility for scaling up or 

down reaction and elution volumes. The purified 

DNA is ideal for all common molecular biology 

procedures, including restriction digestion, 

cloning, and sequencing. 

Features 

Flexible – easy to scale up or down both 

reaction and elution volumes. 

Efficient – greater than 80% DNA recovery. 

Fast – purification takes only 15-20 min. 

Versatile – suitable for DNA fragments as 

short as 100 bp. 

Safe – does not involve phenol extraction. 

M Extracted and Purified DNA Fragments M 

Efficient recovery of individual 100-3000 bp DNA fragments from the 

Thermo Scientific GeneRuler 100 bp Plus DNA Ladder from a 1.7% 

agarose gel using the Silica Bead DNA Gel Extraction Kit. 

M – GeneRuler 100 bp Plus DNA Ladder (#SM0321). 

Applications 

Purification of DNA fragments from agarose 

gels prepared with TAE or TBE buffers. 

Concentration of DNA samples for desalting 

or buffer change. 

Removal of enzymes after PCR, restriction 

digestion, dephosphorylation or labeling 

reactions. 

Removal of unincorporated nucleotides, 

primers and primer-dimers. 

Removal of linkers following ligation. 

Removal of residual salts, phenol, chloroform 

or ethidium bromide. 

Purification of RNA-free plasmid DNA. 


Silica Powder Suspension 

Binding Solution 

Concentrated Wash Buffer 

TBE Conversion Buffer 

 

Note 

 

with UV light when the gel-purified DNA 

fragment will be used in downstream 

cloning reactions. Use a long wavelength 

(360 nm) UV light-box during excision of the 

gel slice, minimize the UV exposure to a few 

seconds or keep the gel on a glass 

or plastic plate. 

References 

1. Vogelstein, B., Gillespie, D., Preparative and analytical 

purification of DNA from agarose, Proc. Natl. Acad. Sci. 

USA, 615-619, 1979. 



Efficiency, Elsevier Trends Journals Technical Tips, Online, 

T40022, 1996. 


gels and educational demonstrations, Anal Biochem., 

240 (1), 17-23, 1996.

Agarase 

#EO0461 100 u (0.5 u/μl) 








Glycogen p.423 


Cut out gel slice containing DNA 

or 

RNA fragment 

Melt agarose 10 min at 70°C 

Equilibrate to 42°C 

Add Agarase 


Add ammonium acetate 

Chill on ice for 5 min 


Add ethanol or isopropanol to 

supernatant 

Incubate 1 h at room temperature; 


Remove supernatant 

Dissolve in TE buffer 


Reference 

1. Yaphe, W., The use of agarase from Pseudomonas 

atlantica in the identification of agar in marine algae 

(Rhodophyceae), Can. J. Microbiol., 3, 987-993, 1957. 

Description 

The Thermo Scientific Agarase allows for 

gentle yet efficient recovery of DNA or RNA 

fragments from low melting point agarose by 

digesting the agarose polysaccharide core into 

oligosaccharides (1). The recovered nucleic 

acids can be directly used for amplification, 

cloning, sequencing, etc. 

Features 

Gentle – recovers intact DNA and RNA. 

High recovery yields – >90% of the starting 

DNA is recovered. 

Efficient – ideal for both long (>30 kb) and 

short (

320 


DNA isolation Cat. # Size Applications Page 

Proteinase K (recombinant), PCR grade, 

~600 u/ml 

Removal of DNA Cat. # Size Supplied with Applications Page 

DNase I, RNase-free, 1 u/μl 


EN0521 1000 u 

EN0525 1000 u 

DNase I, RNase-free, 50 u/μl EN0523 HC, 1000 u 

Exonuclease I, 20 u/μl 

10X Reaction buffer with MgCl 2 1 ml 



10X Reaction buffer w/o MnCl 2 1 ml 

100 mM MnCl 2 1 ml 




EN0581 4000 u 10X Reaction buffer 1 ml 

EN0582 20000 u 10X Reaction buffer 5x1 ml 


» 4.6. Removal of template DNA after in vitro transcription 

» 4.7. Removal of genomic DNA from RNA preparations 

» 4.8. PCR product clean-up prior to sequencing 

 

 

in vitro transcription. 

 

RT-PCR. 

 

removal from PCR mixture for direct 

sequencing. 

 

containing a 3’-hydroxyl terminus 

from nucleic acid mixtures. 

Removal of RNA Cat.# Size Applications Page 

RNase A, DNase and protease-free, 

10 mg/ml 

EN0531 10 mg 

262 

RNase T1, 1000 u/μl 

RNase A/T1 Mix, 2 mg/ml of RNase A, 

5000 u/ml of RNase T1 

RNase I, (10 u/μl) 

EO0491 

EO0492 

1 ml 

20 mg/ml 

5X1 ml 

20 mg/ml 

EN0541 

EN0542 

10,0000 u 

50,0000 u 

263 

EN0551 1 ml 

 

264 

EN0601 1000 u 

EN0602 5000 u 

 

 

 

or cell lines. 


» 4.1. Purification of genomic DNA from mouse tail with Proteinase K 

» 4.2. Purification of DNA from cultured eukaryotic cells with Proteinase K 

270 

p.322 

p.322 

255 

255 

258 

p.323 

p.323 

p.323 

265

PCR clean-up Cat. # Size Supplied with Applications Page 


Alkaline Phosphatase, 1 u/μl 

Exonuclease I, 20 u/μl 

Nucleic acid precipitation Cat. # Size Applications Page 

Glycogen, molecular biology grade, 20 mg/ml 

Glycogen, RNA grade, 20 mg/ml 

R0561 

R0551 

2X 0.25 ml 

2X 0.1 ml 

423 

3 M Sodium Acetate Solution, pH 5.2, 


R1181 5X 1 ml 

 

421 


» 4.5. Nucleic acid precipitation from diluted solutions with Glycogen p.323 

Nucleic acid separation Cat. # Size Applications Page 


TopVision Low Melting Point Agarose R0801 25 g 

EF0651 1000 u 10X FastAP buffer 2x1.5 ml 

EF0652 5X 1000 u 10X FastAP buffer 10x1.5 ml mixture prior to sequencing of PCR 

EF0654 300 u 10X FastAP buffer 1.5 ml products. 

EN0581 4000 u 10X Reaction buffer 1 ml 

EN0582 20000 u 10X Reaction buffer 5x1 ml 

R0491 100 g 

 

R0492 500 g 

 

 

 

 

removal from PCR mixture for direct 

sequencing. 

 

containing a 3’-hydroxyl terminus 

from nucleic acid mixtures. 


» 4.8. PCR product clean-up prior to sequencing p.323 


» 4.3. Casting high quality agarose gels p.322 

» 4.4. Recovery of DNA from LM agarose gels with Agarase p.322 

242 

258 

427 


322 


4.1. Purification of genomic DNA 

from mouse tails with Proteinase K 

1. Place a 0.5-1 cm sample of mouse tail into 

a 1.5 ml microcentrifuge tube. 

2. Add 500 μl of lysis buffer (50 mM KCl, 


0.45% NP-40, 0.45% Tween-20). 

3. Add 2.5 μl of Proteinase K, 20 mg/ml 

(#EO0491) and mix briefly. 

4. Incubate overnight at 55°C. 

Optional. Incubate for an additional 1 h 

at 65°C. 

5. Vortex and spin 10 seconds at 13,000 rpm 

to collect cell debris. 

6. Use 1 μl from the upper part of the 

supernatant per 50 μl of PCR mix. 

4.2. Purification of DNA from 

cultured eukaryotic cells with 

Proteinase K 

The following protocol is used for for 1-2X 10 6 

cells and typically generates 7-15 μg DNA. 

1. Centrifuge for 5 min at 3000 rpm in a 

microcentrifuge to collect the cells. 

2. Wash the pellet twice with PBS 

(137 mM NaCl, 27 mM KCl, 100 mM 

Na 2HPO 4, 2 mM K 2HPO 4, pH 7.4). 

3. Resuspend the pellet in 0.5 ml of lysis buffer 

(10 mM Tris-HCl, (pH 8.5), 5 mM EDTA, 

200 mM NaCl, 0.2% SDS). 

Incubate at 60°C for 5 min. 

4. Add 2.5 μl of Proteinase K (#E00491) and 

5 μl of RNase A/T1 Mix (#EN0551), mix. 

Incubate at 60°C for 1 h. 

5. Add 250 μl of 5 M NaCl, mix and incubate 

on ice for 5 min to precipitate protein. 

6. Centrifuge for 15 min at 10,000 rpm in a 

microcentrifuge. 

7. Transfer supernatant to a fresh tube. Add 

an equal volume of isopropanol and mix to 

precipitate the DNA. 

Optional. Incubate at -20°C for up to 60 min 

to increase the DNA yield. 

8. Centrifuge for 10 min at 10,000 rpm in a 

microcentrifuge. 

9. Discard the supernatant and wash the pellet 

with 1.2 ml of 70% cold ethanol. 

10. Air-dry the pellet*. Dissolve the pellet in 

Water, nuclease-free (#R0581) or TE buffer. 

* Do not use a vacuum dryer or let the pellet dry 

completely. Overdried genomic DNA has low solubility. 


4.3. Casting high quality 

agarose gels 

 

prepare the gel and to run electrophoresis. 

 

ethidium bromide (0.5 μg/ml) to both the 

electrophoresis buffer and gel. Wear gloves 

when handling ethidium bromide. 

 

supercoiled or relaxed plasmid DNA, 

do not include ethidium bromide in the 

electrophoresis buffer or gel. The gel should 

be stained only after electrophoresis. 

 

use a flask of a volume at least three times 

larger than that of the solution to avoid 

solution loss due to boiling. 

Procedure: 

1. Weigh out the required amount (depending 

on the gel percentage) of agarose into an 

Erlenmeyer flask and add the appropriate 

volume of either 1X TBE or 1X TAE buffer, 

swirl to mix. 

2. Weigh the flask with the solution. 

3. For high percentage (3-5%) agarose gels: 

add a 10-20% excess amount of 

distilled water. 

4. Boil the mixture in a microwave oven 

at medium power until the agarose is 

completely molten, swirl several times while 

solution is still boiling. 

To prepare the highest quality agarose gels 

of any percentage, an additional 3-5 min 

boiling after complete melting of the 

agarose is recommended. 

5. Weigh the flask again and, if necessary, add 

hot distilled water to restore the evaporated 

water (or continue boiling) to obtain the 

correct percentage agarose gel. 

Optional. Add ethidium bromide to a final 

concentration of 0.5 μg/ml, mix well and 

heat the mixture for an additional minute 

without boiling. 

6. Cool the solution to 65-70°C. Pour carefully 

on a clean casting plate with an appropriate 

comb. If bubbles appear, push them carefully 

away to the sides with a pipette tip. 

7. Allow the gel to solidify for about 30 min 

before using. Low percentage low-melting 

point agarose gels can only be 

solidified at 4°C. 

8. Immerse the gel into the desired 

electrophoresis buffer and load samples. 

9. After electrophoresis the gel can be stained 

with ethidium bromide, SYBR Green I or by 

any other staining technique. 

Warning: hot agarose solution should be 

handled very carefully. 

4.4. Recovery of DNA from LM 

agarose gels with Agarase 

1. Perform electrophoresis of DNA in a low 

melting point (LM) agarose (#R0801) gel 

prepared in TAE (#B49), 0.5X TBE, 

TBE (#B52) or TPE buffer. Stain the gel 

with ethidium bromide. 

2. Cut out the desired DNA band from the 

agarose gel with a clean scalpel under 

UV light*. Cut out only as much agarose 

as necessary. (The bottom of the excised 

agarose slice is free of DNA and should be 

removed). 

3. Determine the weight of the slice. To 

facilitate melting, cut gel slices larger than 

200 mg into smaller pieces. 

4. Incubate the tube at 70°C for approx. 10 min. 

Ensure that the agarose is completely molten. 

5. Transfer the tube to a 42°C water bath and 

equilibrate for 5 min. 

6. Add 1 u of Agarase (#EO0461) per 

100 mg (approx. 100 μl) of molten 1% LM 

agarose. Increase the amount of enzyme 

proportionally for higher percentage agarose, 

gently mix and incubate at 42°C for 30 min. 

7. Add ammonium acetate** to a 2.5 M final 

concentration, chill on ice for 5 min. 

8. Centrifuge at 10,000 rpm for 10 min to 

pellet undigested carbohydrates. Transfer 

the supernatant to a clean tube. 

9. Add 2.5 volumes of ethanol or 0.8 volume 

of isopropanol, mix gently and incubate at 

room temperature for 1 h. If DNA fragments 

are smaller than 500 bp or if the DNA 

concentration is lower than 0.05 μg/ml, 

incubate at room temperature for 2 h. 

10. Centrifuge at 10,000 rpm for 15 min, 

remove supernatant and dry the pellet. 

Resuspend the pellet in TE or another 

appropriate buffer for subsequent 

manipulation. 

Note 

* For subcloning of gel-purified DNA fragments, care should 

be taken to avoid DNA damage with UV light. Minimize the 

UV exposure to a few seconds or keep the gel on a glass 

or plastic plate during UV illumination. Alternatively, visible 

dyes can be included in standard agarose gels to visualize 

DNA bands in ambient light (1, 2). 

** Ammonium acetate is recommended rather than other 

salts to avoid co-precipitation of oligosaccharides with 

DNA. 

 

 

therefore use 3 M Sodium Acetate, #R1181, (0.3 M final 

concentration) if T4 Polynucleotide Kinase will be used in 


 

to avoid mechanical shearing. After digestion with Agarase 

(step 6), centrifuge at maximum speed for 10 min to pellet 

undigested carbohydrates. Remove oligosaccharides and 

agarase by dialysis or carry out subsequent manipulations 

with DNA in the digested agarose solution.

4.5. Nucleic acid precipitation 

from diluted solutions with 

Glycogen 

1. Add 1/10 volume of 3 M Sodium Acetate 

(#R1181), 2 M sodium chloride or 5 M 

ammonium acetate to DNA solution. 

2. Add Glycogen (#R0561 for DNA or 

#R0551 for RNA) to a final concentration 

of 0.05-1 μg/μl. Use up to 1 μl of Glycogen 

per 20 μl of solution. 

3. Add 2.5 volumes of ethanol. Mix gently but 

thoroughly. 

4. Incubate for 5 min at room temperature. 

5. Centrifuge the mixture for 10-15 min at 

10,000 rpm. Discard the supernatant. 

6. Rinse the pellet with cold 70% ethanol. Airdry 

the pellet. 

7. DNA: Dissolve the pellet in Water, nucleasefree 

(#R0581) or TE buffer. 

RNA: Dissolve pellet in DEPC-treated Water 

(#R0601). 

4.6. Removal of template DNA 

after in vitro transcription 

1. Add 2 u of DNase I, RNase-free (#EN0521) 

per 1 μg of template DNA directly to the 

transcription reaction mixture. In some 

cases, the amount of enzyme should be 

determined empirically. 

2. Incubate at 37°C for 15 min. 

3. Inactivate DNase I by phenol/chloroform 

extraction. 

References 



Efficiency, Elsevier Trends Journals Technical Tips Online, 

T40022, 1996. 


gels and educational demonstrations, Anal. Biochem., 

240 (1), 17-23, 1996. 



252-256, 2000. 



252-256, 2000. 

5. Werle, E., et al., Convenient single-step, one tube 

purification of PCR products for direct sequencing, Nucleic 

Acids Res., 22, 4354-4355, 1994. 

4.7. Removal of genomic DNA 

from RNA preparations 

1. Add to an RNase-free tube: 

RNA 1 μg 

10X reaction buffer with MgCl2 1 μl 

DNase I, RNase-free (#EN0521) 1 μl (1 u) 

DEPC-treated Water (#R0601) to 10 μl 



3. Add 1 μl of 50 mM EDTA and incubate at 

65°C for 10 min. RNA hydrolyzes when 

heated in the absence of a chelating agent (4). 

4. Use the prepared RNA as a template for 

reverse transcription. 

Note 

 

of RNA. 

RNase Inhibitor (#EO0381), typically at 1 u/μl, 

can also be included into the reaction mixture to prevent 

RNA degradation. 

 

RNase-free. 

4.8. PCR product clean-up prior 

to sequencing 

The clean-up reaction degrades unincorporated 

primers and nucleotides. The resulting PCR 

product is ready to use for sequencing without 

additional purification; e.g., using column 

purification kits. 


PCR mixture (directly after completion of PCR) 5 μl 

Exonuclease I (#EN0581) 0.5 μl (10 u) 


(#EF0651) 

1 μl (1 u) 

2. Mix well and incubate at 37°C for 15 min. 

3. Stop the reaction by heating the mixture at 


Note 

 

DNA sequencing without further purification. 

 

specific PCR products present. 

 

generated by any thermophilic DNA polymerase or 

polymerase mix. 

 

cloning applications. 

4.9. Phenol/chloroform 

extraction and ethanol 

precipitation 

1. Mix sample with 0.5 volume of Trissaturated 

phenol and 0.5 volume of 

chloroform. Sample to phenol/chloroform 

ratio must be 1:1. Centrifuge at 10,000 rpm 

for 5 min at room temperature. 

2. Transfer the upper aqueous phase to a fresh 

tube. Add an equal volume of chloroform 

and mix. Centrifuge at 10,000 rpm for 

5 min at room temperature. Repeat. 

3. Transfer the upper aqueous phase to 

a fresh tube. Add 1/10 the volume of 

3 M Sodium Acetate Solution (#R1181) 

or 2 M sodium chloride. 

4. Add 2.5 volumes of ethanol or an equal 

volume of isopropanol to precipitate DNA. 

5. Incubate the mixture for 30 min at -20°C. 

6. Centrifuge for 10 min at 10,000 rpm. Then 

discard the supernatant and rinse the pellet 

with 70% cold ethanol. 

7. Air-dry the pellet. Dissolve in Water, 

nuclease-free (#R0581) or TE buffer. 

Note 

Use Glycogen (#R0561) to maximize the yield of DNA during 

precipitation (see protocol above). 


324 

The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC. 


Thermo Scientific in vitro 

Transcription Products 


Products .............................................................................................................. 328 

TranscriptAid T7 High Yield Transcription Kit ........................................................... 328 

Replicator RNAi Kit ......................................................................................................329 

T7 RNA Polymerase .....................................................................................................330 

T3 RNA Polymerase and SP6 RNA Polymerase .......................................................... 331 

Phi6 RNA Replicase......................................................................................................332 



5.1. DNA template preparation for in vitro transcription ......................................................335 

5.2. Avoiding RNase contamination ..................................................................................335 

5.3. In vitro transcription ..................................................................................................335 

5.4. Synthesis of radiolabeled RNA probes of high specific activity .....................................335 

5.5. Purification of RNA transcripts ..................................................................................336 

5.6. Evaluation of transcription reaction products ..............................................................336 

Troubleshooting Guide ........................................................................................ 337 


326 


Applications Procedures Products to use Cat. # Page 

Generation of template 

DNA for in vitro transcription 

High yield in vitro 

transcription up to 200 μg 

RNA per 20 μl reaction 

Conventional 

in vitro transcription 

>10 μg RNA 

per 20 μl reaction 

RNA amplification 


Cloning into vectors containing 

an RNA polymerase promoter 

Generation of PCR products 

Plasmid purification 

Linearization of plasmid DNA 

Blunting of 3’-overhangs 

Unlabeled RNA and 

high specificity non-radiolabeled 

RNA probes 

Unlabeled RNA and 

high specificity radiolabeled RNA probes 

First strand cDNA synthesis 

CloneJET PCR Cloning Kit (T7 promoter) K1231/2 279 

InsTAclone PCR Cloning Kit* (T7 promoter) K1213/4 281 

pTZ19R (T7 promoter) SD0141 445 

FastDigest Restriction Enzymes FDXXXX 2 

Conventional Restriction Enzymes ERXXXX 77 

FastAP Thermosensitive Alkaline Phosphatase EF0651/2/4 242 

Rapid DNA Ligation Kit K1422/3 288 

T4 DNA Ligase EL0014 239 

See complete portfolio for Thermo Scientific PCR and RT-PCR products 





FastDigest Restriction Enzymes FDXXXX 2 

Conventional Restriction Enzymes ERXXXX 77 

T4 DNA Polymerase EP0061/2 249 

dNTP Mix, 2 mM each R0241/2 408 

TranscriptAid T7 High Yield Transcription Kit K0441 328 

Aminoallyl-UTP, 50 mM R1091 414 

Pyrophosphatase, Inorganic EF0221 272 

T7 RNA Polymerase EP0111/2/3 330 


SP6 RNA Polymerase EP0131/3 331 

Pyrophosphatase, Inorganic EF0221 272 

RiboLock RNase Inhibitor EO0381/2/4 271 

NTP Set, 100 mM R0481 411 

ATP, 100 mM R0441 411 

GTP, 100 mM R0461 412 

CTP, 100 mM R0451 412 

UTP, 100 mM R0471 412 

DEPC-treated Water R0601/3 420 



RiboLock RNase Inhibitor EO0381/2 271 

dNTP Mix, 10 mM each R0191/2/3 408 

DEPC-treated Water R0601/3 420 

Second strand cDNA synthesis 


RNase H 

EP0041/2 

EN0201/2 

246 

266 

High yield in vitro transcription TranscriptAid T7 High Yield Transcription Kit K0441 328 

* not available in USA. (continued on next page)

Applications Products to use Cat. # Page 

Complete kit for production of dsRNA from 

any target DNA template 

Replicator RNAi Kit F-610 329 

Generation of ssRNA from DNA template 



ds RNA Synthesis 


Synthesis of complementary RNA strand 

from the 3’ terminus of a ssRNA 

Phi6 RNA Replicase F-611S/L 332 

Cleavage of dsRNA to small interfering 

RNA (siRNA) 

PowerCut Dicer F-602S/L 267 

Labeling of RNA See chapter Molecular Labeling & Detection 

Removal of template DNA 

Precipitation of RNA 

Analysis of RNA 

RNA standards 

Agarose 

Blotting and detection Biotin detection 

DNase I, RNase-free EN0521/3/5 255 

0.5 M EDTA, pH 8.0 R1021 421 

3 M Sodium Acetate Solution, pH 5.2 R1181 421 

Glycogen, RNA grade R0551 423 

RiboRuler Low Range RNA Ladder SM1831 

RiboRuler Low Range RNA Ladder, ready-to-use 

RiboRuler High Range RNA Ladder 

SM1833 

SM1821 

386 

RiboRuler High Range RNA Ladder, ready-to-use SM1823 

Phi6 dsRNA F-630 387 

TopVision Agarose R0491/2 

TopVision Low Melting Point Agarose R0801 

Electrophoresis buffers 



B52 

B49 

371 

Loading dye solution 2X RNA Loading Dye R0641 388 

Biotin Chromogenic Detection Kit K0661 345 

BCIP-T 

NBT 

R0821/2 

R0841/2 

426 

427 


328 

Products 

TranscriptAid T7 High Yield Transcription Kit 

#K0441 for 50 rxns (of 20 μl) 



PCR Cloning Kit p.281 

pTZ19R DNA p.292 


Aminoallyl-UTP p.414 

NTP Set p.411 

RiboLock 

 

 


2X RNA Loading Dye p.388 





Alkaline Phosphatase p.242 


μg of RNA 

250 

200 

150 

100 

6000 b 

1000 b 

50 

500 b 

0 

0 30 min 1 h 2 h 4 h 

Time course of RNA synthesis. 

500 b, 1000 b and 6000 b RNA transcripts 

were generated with TranscriptAid T7 High Yield 

Transcription Kit. 1 μg of DNA template was used in 

20 μl reactions. Yields of RNA were determined at 

different transcription reaction time points using an 

Agilent 2100 Bioanalyzer. 


Time 


» 5.1. DNA template preparation for in vitro 


» 5.2. Avoiding RNase contamination p.335 

» 5.5. Purification of RNA transcripts p.336 

Description 

The Thermo Scientific TranscriptAid T7 High 

Yield Transcription Kit is designed for high 

yield in vitro transcription from DNA templates 

containing a T7 RNA Polymerase promoter, 

including linearized plasmids or PCR products. 

The kit contains reagents for 50 reactions of 

20 μl. Each reaction yields up to 200 μg RNA 

from 1 μg of template in 2 hours. The reaction 

can be scaled up to produce milligram amounts 

of full-length RNA. 

The kit can be used to produce both long and 

short transcripts for applications that require 

large yields of RNA. All necessary reagents 

for transcription are included, as well as the 

Thermo Scientific RiboRuler High Range RNA 

Ladder for sizing and quantification. 

Features 

Exceptionally high yields – up to 200 μg 

in 2 hours. 

Versatile – ideal for both short and long RNA 

transcripts. 

Milligram amounts of RNA in a single, 

scaled-up reaction. 

Flexible – generates unlabeled, labeled or 

capped RNA. 

RiboRuler RNA Ladder supplied with kit for 

sizing and quantification. 

μg of RNA 

160 

140 

120 

100 

80 

60 

40 

20 

0 

100 b 

50 b 

0 2 4 

Time, hours 

6 

Time course of short RNA synthesis. 

50 b and 100 b RNA transcripts were generated with 

TranscriptAid T7 High Yield Transcription Kit in 0-8 

hour reaction times. RNA yield was evaluated with the 

Agilent 2100 Bioanalyzer. 

8 

Applications 


In vitro translation. 

Generation of hybridization probes for: 

– microarrays, 

– in situ hybridization, 

– blotting. 

RNase protection assays. 

RNA binding protein assays. 

Antisense RNA and RNAi. 

RNA amplification. 

Microinjection studies. 


Enzyme Mix 

Reaction Buffer 

 

 

 

 

 

 

 

DEPC-treated Water 

 

High Range RNA Ladder, 

ready-to-use 

 

 

Thermo Scientific vendor A vendor B 

M 1 2 3 4 5 6 7 8 9 M 

High yield of full length RNA transcripts. 

500 b, 1000 b and 6000 b RNA transcripts were 

generated using high yield in vitro transcription 

reactions containing 1 μg of DNA template. Each 

20 μl reaction was incubated for 2 hours at 37°C. The 

transcription products were diluted 80-fold and 6 μl 

was run on a 1% agarose/TAE gel supplemented with 

0.5 μg/ml ethidium bromide. 

M – RiboRuler High Range RNA Ladder, 

ready-to-use (#SM1823). 

1, 4, 7 – 500 b RNA transcripts. 


3, 6, 9 – 6000 b RNA transcripts.

Replicator RNAi Kit 

#F-610 for 40 rxns (of 50 μl) 


 

NTP Set p.411 

dNTPs p.408 



 

 


PowerCut Dicer p.267 

References 

1. Makeyev, E.V., Bamford, D.H., Replicase activity of 

purified recombinant protein P2 of double-stranded RNA 

bacteriophage 6, EMBO J., 19, 124-133, 2000. 

2. Makeyev, E.V., Bamford, D.H., The polymerase subunit of 

a dsRNA virus plays a central role in the regulation of viral 

RNA metabolism, EMBO J., 19, 6275-6284, 2000. 

3. Laurila, M.R.L. et al., Bacteriophage 6 RNA-dependent 

RNA Polymerase, J. Biol. Chem., 277, 

17117-17124, 2002. 

4. Calegari, F. et al., Tissue-specific RNA interference in 

postimplantation mouse embryos with endoribonucleaseprepared 

short interfering RNA, Proc. Natl. Acad. Sci. USA, 

99, 14236-14240, 2002. 

5. Myers, J.W. et al., Recombinant dicer efficiently converts 

large dsRNAs into siRNAs suitable for gene silencing, Nat. 

Biotechnol., 21, 324-328, 2003. 

6. Yang D. et al., Short RNA duplexes produced by hydrolysis 

 

interference in mammalian cells, Proc. Natl. Acad. Sci. 

USA, 99, 9942-9947, 2002. 

Description 

The Thermo Scientific Replicator RNAi Kit 

is a complete kit for producing milligram 

quantities of double-stranded RNA. Perfectly 

duplexed RNA produced with this kit can be 

further processed into siRNA which is the key 

component in RNAi-mediated gene silencing 

experiments. The DNA template is created by 

PCR amplification using Phusion High-Fidelity 

DNA Polymerase to ensure the highest possible 

 

DNA-dependent RNA Polymerase transcribes 

single-stranded RNA copies from the DNA 

template molecule. Subsequently, the ssRNA 

molecules are directly converted to dsRNA 

by Phi6 RNA-dependent RNA polymerase in 

the same reaction mixture. One kit contains 

materials for producing up to 2.5 mg of pure 

double-stranded RNA in a 

2 ml reaction volume. The high yield of doublestranded 

RNA obtained with the system is due 

to a unique combination of the complementary 

polymerase activities in carefully optimized 

reaction conditions. 

Applications 

Production of double-stranded RNA in 

milligram quantities for gene silencing 

experiments based on the RNAi mechanism. 

Production of a dsRNA marker fragment with 

a specific size. 

Features 

Convenient – no RNA hybridization step in 

the reaction protocol. 

Efficient – high yields of perfectly duplexed 

RNA are produced. 

No smeared product resulting from 

RNA secondary structure formation or 

misannealing on agarose gels. 

dsRNA fragments up to 20 kb can be 

amplified. 


 

 

 

 

 

 

 

 

 

2 Solution 

 

 

M 1 2 3 4 5 M 

Various gene-specific dsRNA fragments generated using 

the Thermo Scientific Replicator RNAi Kit. 

PCR-amplified fragments of various sizes were used as template 

DNA. 0.1 μl from each reaction mixture was loaded 

on an agarose gel. 

M 

1 - dsRNA specific to 150bp sequence of lambda DNA 

2 - dsRNA specific to 210 bp sequence of Furin gene 

3 - dsRNA specific to 281 bp sequence of Erk1/2 kinase gene 

4 - dsRNA specific to 430 bp sequence of Caspase-3 gene 

5 - dsRNA specific to 864 bp sequence of lambda DNA 


330 

T7 RNA Polymerase 

#EP0111 5000 u (20 u/μl) 


5X Transcription Buffer 1.25 ml 

#EP0112 5x5000 u (20 u/μl) 

#EP0113 HC, 25000 u (>100 u/μl) 

Both Supplied with: 

5X Transcription Buffer 5x1.25 ml 



RiboLock 

 


NTP Set p.411 


 

Glycogen, RNA grade p.423 

Agarase p.319 

0.5 M EDTA, pH 8.0 p.421 









Description 

The Thermo Scientific T7 RNA Polymerase is 

a DNA-dependent RNA polymerase with strict 

specificity for its respective double-stranded 

promoter. The enzyme catalyzes the 5’3’ 

synthesis of RNA on either single-stranded DNA 

or double-stranded DNA downstream from the 

promoter. 

Feature 

modified nucleotides (e.g., 

aminoallyl-, biotin-, fluorescein-, digoxigeninlabeled 

nucleotides). 

Applications 

Synthesis of unlabeled and labeled RNA that 

can be used: 

– for hybridization (1), in vitro RNA 

translation (2); 

– as antisense RNA (3), siRNA (4), substrate 

in RNase protection assays (5) and 

template for genomic DNA sequencing (6); 

– in studies of RNA secondary structure 

and RNA-protein interactions (7), 

RNA splicing (8). 

Source 

E.coli cells with a cloned gene encoding the 

T7 RNA Polymerase. 



Concentration 

20 u/μl 

>100 u/μl, HC 


One unit of the enzyme incorporates 1 nmol of 

AMP into a polynucleotide fraction (adsorbed on 

DE-81) in 60 minutes at 37°C. 


mixture: 40 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 10 mM DTT, 2 mM spermidine, 0.5 mM of each 

NTP, 0.6 MBq/ml [ 3H]-ATP, 20 μg/ml plasmid 

DNA containing the T7 promoter sequence. 




5 mM DTT, 0.1 mg/ml BSA, 

0.03% (v/v) ELUGENT Detergent, 50% (v/v) 

glycerol. 

5X Transcription Buffer 

200 mM Tris-HCl (pH 7.9 at 25°C), 30 mM MgCl2, 50 mM DTT, 50 mM NaCl, 10 mM spermidine. 


 

reduced by 50% at NaCl or KCl concentration 

above 150 mM. 

y heating at 70°C for 10 min or 


Note 

 

-15 -10 -5 +1 +5 

T7 TAATACGACTCACTATAGGGAGA 

Position +1 indicates the first nucleotide 

incorporated into RNA during transcription. 

Only bases at positions +1 through +3 are 

critical for transcription, and they must be 

a G and 2 purine bases, respectively (9). 

References 

1. Melton, D.A., et al., Efficient in vitro synthesis of biologically 

active RNA and RNA hybridization probes from plasmids 

containing a bacteriophage SP6 promoter, Nucleic 

Acids Res.,12, 7035-7056, 1984. 

2. Krieg, P.A., Melton, D.A., Functional messenger RNAs are 

produced by SP6 in vitro transcription of cloned cDNAs, 


 

messenger RNA translation in vivo, Proc. Natl. Acad. Sci. 

USA, 82, 144-148, 1985. 

4. Bernstein, E., et al., Role for bidentate ribonuclease in the 

initiation step of RNA interference, Nature, 409, 

363-366, 2001. 

5. Peebles, C.L., et al., A self-splicing RNA excises an intron 

lariat, Cell, 44, 213-223, 1986. 

6. Church, G.M., Gilbert, W., Genomic sequencing, Proc. Natl. 

Acad. Sci. USA, 81, 1991-1995, 1984. 

7. Witherell, G.W., et al., Cooperative binding of R17 coat 

protein to RNA, Biochemistry, 29, 11051-11057, 1990. 

8. Krainer, A.R., et al., Normal and mutant human -globin 

pre-mRNAs are faithfully and efficiently spliced in vitro, 

Cell, 36, 993-1005, 1984. 

9. Jorgensen, E.D., et al., Specific contacts between the 

bacteriophage T3, T7, and SP6 RNA polymerases and 

their promoters, J. Biol. Chem., 266, 645-651, 1991.

T3 RNA Polymerase and SP6 RNA Polymerase 

T3 RNA Polymerase 

#EP0101 2000 u (20 u/μl) 



#EP0102 10000 u (20 u/μl) 

#EP0103 HC, 10000 u (>100 u/μl) 



SP6 RNA Polymerase 

#EP0131 2000 u (20 u/μl) 



#EP0133 HC, 5000 u (>100 u/μl) 




RiboLock 

 


NTP Set p.411 


 

Glycogen, RNA grade p.423 

Agarase p.319 

0.5 M EDTA, pH 8.0 p.421 


References 

1. Melton, D.A., et al., Efficient in vitro synthesis of 

biologically active RNA and RNA hybridization probes 

from plasmids containing a bacteriophage SP6 promoter, 

Nucleic Acids Res.,12, 7035-7056, 1984. 

2. Krieg, P.A., Melton, D.A., Functional messenger RNAs are 

produced by SP6 in vitro transcription of cloned cDNAs, 


 

messenger RNA translation in vivo, Proc. Natl. Acad. Sci. 

USA, 82, 144-148, 1985. 

4. Bernstein, E., et al., Role for bidentate ribonuclease in the 

initiation step of RNA interference, Nature, 409, 

363-366, 2001. 

5. Peebles, C.L., et al., A self-splicing RNA excises an intron 

lariat, Cell, 44, 213-223, 1986. 

6. Church, G.M., Gilbert, W., Genomic sequencing, Proc. Natl. 

Acad. Sci. USA, 81, 1991-1995, 1984. 

7. Witherell, G.W., et al., Cooperative binding of R17 coat 

protein to RNA, Biochemistry, 29, 11051-11057, 1990. 

8. Krainer, A.R., et al., Normal and mutant human -globin 

pre-mRNAs are faithfully and efficiently spliced in vitro, 

Cell, 36, 993-1005, 1984. 

9. Jorgensen, E.D., et al., Specific contacts between the 

bacteriophage T3, T7, and SP6 RNA polymerases and 

their promoters, J. Biol. Chem., 266, 645-651, 1991. 

Description 

Thermo Scientific T3 RNA and SP6 RNA 

Polymerases are DNA dependent RNA 

polymerases with strict specificity for their 

respective double-stranded promoters.The 

enzymes catalyze the 5’3’ synthesis of RNA 

on either single-stranded DNA or doublestranded 

DNA downstream from the promoter. 

Features 

 

(e.g., minoallyl-, biotin-, fluorescein-, 

digoxigeninlabeled nucleotides). 

Applications 

Synthesis of unlabeled and labeled RNA that 

can be used: 

– for hybridization (1), in vitro RNA 

translation (2); 

– as aRNA (3), siRNA (4), substrate in RNase 

protection assays (5) and template for 

genomic DNA sequencing (6); 

– in studies of RNA secondary structure 

and RNA-protein interactions (7), RNA 

splicing (8). 

Source 

E.coli cells with a cloned gene encoding the 

T3 RNA Polymerase or SP6 RNA Polymerase. 


T3 RNA Polymerase – 99 kDa monomer. 

SP6 RNA Polymerase – 99 kDa monomer. 

Concentration 

20 u/μl 

>100 u/μl, HC 


One unit of the enzyme incorporates 1 nmol of 

AMP into a polynucleotide fraction (adsorbed on 

DE-81) in 60 minutes at 37°C. 


mixture: 40 mM Tris-HCl (pH 8.0), 6 mM MgCl2, 10 mM DTT, 2 mM spermidine, 0.5 mM 

of each NTP, 0.6 MBq/ml [ 3H]-ATP, 20 μg/ml 

plasmid DNA containing the T3 or SP6 

promoter sequence. 


The enzymes are supplied in: 


5 mM DTT, 0.1 mg/ml BSA, 

0.03% (v/v) ELUGENT Detergent, 


5X Transcription Buffer 


30 mM MgCl2, 50 mM DTT, 50 mM NaCl, 

10 mM spermidine. 


 

reduced by 50% at NaCl or KCl concentration 

above 250 mM (for T3 RNA Polymerase) or 

above 150 mM (for SP6 RNA Polymerase). 

 


Note 

 

-15 -10 -5 +1 +5 

T3 AATTAACCCTCACTAAAGGGAGA 

SP6 ATTTAGGTGACACTATAGAAGNG 

Position +1 indicates the first nucleotide 

incorporated into RNA during transcription. 

Only bases at positions +1 through +3 are 

critical for transcription, and they must be 

a G and 2 purine bases, respectively (9). 








332 

Phi6 RNA Replicase 

#F-611S 


60 u (1 u/μl) 

10x Reaction Buffer 1.5 ml 

50 mM MnCl2 500 μl 

#F-611L 


300 u (1 u/μl) 

10x Reaction Buffer 1.5 ml 

50 mM MnCl2 500 μl 


 

NTP Set p.411 



 

 



Description 

The Thermo Scientific Phi6 RNA Replicase 

is a modified version of the protein P2 from 

bacteriophage Phi6. This RNA-dependent 

RNA polymerase catalyzes the synthesis of a 

full-length complementary RNA strand initiating 

from the 3’ terminus of a single-stranded 

RNA. The polymerase does not require any 

oligonucleotide primer for the initiation. Due to 

a unique modification, the Phi6 RNA Replicase 

displays relatively low template specificity and 

is therefore capable of replicating a wide variety 

of RNA templates, as well as denatured DNA 

which contains a recognition sequence for the 

Phi6 RNA Replicase at its 3’ terminus. 

Applications 

Replication of ssRNA to dsRNA form. 

Source 

E.coli cells with a cloned gene of modified 

version of protein P2 from bacteriophage6. 


75 kDa 

Concentration 

1 u/μl 



incorporation of 1 nmole of UTP into 

an acid-insoluble polynucleotide fraction in 

20 minutes at 32°C. 


mixture: 50 mM Tris-acetate (pH 8.75 at 21°C), 

50 mM NH4-acetate, 1.5 mM MnCl2, 10 % DMSO, 1 mM UTP; 1 μg poly (rA) and 

1 μCi 3H-UTP per 30 μl reaction volume. 




0.1 mM EDTA, 100 mM NaCl, 

0.1 % Triton X-100 and 50 % glycerol. 



500 mM NH4-acetate. Inhibition and Activation 

 

References 

1. Makeyev, E.V., Bamford, D.H., Replicase activity of 

purified recombinant protein P2 of double-stranded RNA 

bacteriophage 6, EMBO J., 19, 124-133, 2000. 

2. Makeyev, E.V., Bamford, D.H., The polymerase subunit of 

a dsRNA virus plays a central role in the regulation of viral 

RNA metabolism, EMBO J., 19, 6275-6284, 2000. 

3. Laurila, M.R.L. et al., Bacteriophage 6 RNA-dependent 

RNA polymerase, J. Biol. Chem., 277, 

17117-17124, 2002.


Ribonuclease inhibition Cat. # Size Applications Page 

RiboLock RNase Inhibitor, 40 u/μl 

Transcription enhancement Cat. # Size Supplied with Applications Page 

Pyrophosphatase, Inorganic*, 

(from yeast), 0.1 u/μl 

EO0381 2500 u 

EO0382 4x2500 u 

EO0384 24x2500 u 

EF0221 10 u Storage (Dilution) buffer 1 ml 

* Use of this product in certain applications may be covered by patents and may require a license. 

in vitro 

transcription reactions by 

reducing the inhibitory effect of 

pyrophosphates. 

RNA ligation Cat. # Size Supplied with Applications Page 

T4 RNA Ligase, 10 u/μl EL0021 1000 u 

 

to 55°C under a wide range of reaction conditions. 


» 5.3. In vitro transcription 

» 5.4. Synthesis of radiolabeled RNA probes of high specific activity 

RNA transcript clean-up Cat. # Size Supplied with Applications Page 

DNase I, RNase-free, 1 u/μl EN0521 1000 u 


DNase I, RNase-free, 50 u/μl EN0523 HC, 1000 u 

Agarase, 0.5 u/μl EO0461 100 u – 

RNA transcript sizing 

and quantification 

RiboRuler Low Range RNA Ladder SM1831 5x20 μl 


ready-to-use 

SM1833 5x40 μl 

RiboRuler High Range RNA Ladder SM1821 5x20 μl 


ready-to-use 

SM1823 5x40 μl 



10X Reaction buffer with MgCl2 1 ml 

10X Reaction buffer w/o MnCl2 1 ml 

100 mM MnCl 2 

1 ml 




10X Reaction buffer 0.2 ml 

10 mM ATP solution 0.2 ml 

1 mg/ml BSA solution 0.2 ml 

in 

vitro transcription reactions. 

 

low melting point (LM) agarose gels. 

 

RNA 3’-end labeling with cytidine 

3’, 5’-bis [- 32 P] phosphate. 

271 

p.335 

p.335 


» 4.6. Removal of template DNA after in vitro transcription p.323 

Cat. # Size Supplied with Applications Page 

2X RNA Loading Dye 1 ml 

 

native or denaturing gels. 

 


» 8.1. General recommendations for RNA electrophoresis p.390 

» 8.2. Preparation of RNA ladders for electrophoresis p.390 

272 

255 

319 

240 

386 


334 

Nucleotides Cat. # Size Applications Page 

NTP Set, 100 mM R0481 4x25 μmol 

411 

ATP, 100 mM 

CTP, 100 mM 

GTP, 100 mM 

R0441 

R0451 

R0461 

25 μmol 

25 μmol 

25 μmol 


 

 

 

411 

412 

412 

UTP, 100 mM R0471 25 μmol 412 

Aminoallyl-UTP, 50 mM R1091 2.5 μmol 

Reaction setup and termination Cat. # Size Applications Page 



R0581 

R0582 

R0603 

R0601 

5x1 ml 

30 ml 

5x1 ml 

30 ml 

 

420 

420 

0.5 M EDTA, pH 8.0 R1021 5x1 ml 421 

RNA electrophoresis Cat. # Size Applications Page 

2X RNA Loading Dye R0641 1 ml 388 

50X TAE Buffer B49 1 litre 

10X TBE Buffer B52 1 litre 

 

 

371 




in vitro 

transcription. 

RNA precipitation Cat. # Size Applications Page 

Glycogen, RNA grade R0551 2x0.1 ml 423 

3 M Sodium Acetate Solution, pH 5.2 R1181 1 ml 421 


» 5.5. Purification of RNA transcripts p.336 

R0491 100 g 

 

R0492 500 g 

 

 

 


» 8.1. General recommendations for RNA electrophoresis p.390 

» 5.6. Evaluation of transcription reaction products p.336 

414 

427


5.1. DNA template preparation 

for in vitro transcription 

Double-stranded linear DNA with blunt or 

5’-protruding ends can be used as template 

for in vitro transcription. Linearized plasmid 

DNA, PCR products or cDNA can be used as 

templates for transcription if they contain a 

double-stranded RNA polymerase promoter 

region in the correct orientation. 

Consensus promoter sequences of different 

RNA Polymerases: 

-15 -10 -5 +1 

T7 TAATACGACTCACTATAGGG 

T3 AATTAACCCTCACTAAAGGG 

SP6 ATTTAGGTGACACTATAGAA 

G will be the first base (+1) of the RNA transcript 

The synthesis of sense or antisense RNA 

transcripts depends on the orientation of 

the promoter with respect to the target 

sequence. The target sequence must be 

placed downstream of the promoter for sense 

RNA and must be inverted for antisense RNA 


Plasmid Templates 

Quality 

Plasmid DNA quality affects transcription 

yield and the integrity of synthesized RNA. 

The greatest transcription yields are achieved 

with high purity plasmid templates. Plasmids 

purified by common laboratory methods can 

be used if the DNA is free of contaminating 

RNases, SDS, EDTA, proteins, salts* and RNA. 

DNA should have a A260/A280 ratio of 1.8-2.0. 

The GeneJET Plasmid Miniprep Kit (#K0502) 

generates high purity plasmid DNA suitable for 


* T7 and SP6 RNA Polymerases are inhibited by ~50% at 

NaCl or KCl concentrations above 150 mM and T3 RNA 

Polymerase – at above 250 mM. 

Linearization 

To produce RNA transcripts of a defined 

length, plasmid DNA is linearized by restriction 

digestion downstream of the insert. Restriction 

enzymes which generate blunt ends or 

5’-overhangs are preferred. 3’-overhangs 

have been reported to generate spurious 

transcripts (1) and should therefore be avoided. 

3’-overhangs can be blunted by T4 DNA 

Polymerase (#EP0061) prior to transcription. 

Due to the high processivity of RNA 

polymerases, circular plasmid templates 

generate long heterogeneous RNA transcripts 

in higher quantities than linear templates. 

Therefore, it is important to completely linearize 

plasmid DNA to ensure efficient synthesis 

of defined length transcripts. See also 

“Troubleshooting Guide for DNA Digestion” on 

 

gel purify the linearized DNA template prior to 

transcription reactions. After linearization, it is 

recommended to purify the DNA template using 

GeneJET PCR Purification Kit (#K0701) or by 

phenol/chloroform extraction: 

1. Add 1/10 volume of 3 M Sodium Acetate 

Solution (#R1181) to the DNA. 

2. Mix thoroughly. 

3. Extract with an equal volume of a 1:1 

phenol/chloroform mixture, and then twice 

with an equal volume of chloroform. Collect 

the aqueous phase and transfer to a new 

tube. 

4. Precipitate the DNA by adding 2 volumes 

 

least 30 min and collect the pellet by 

centrifugation. 

5. Remove the supernatant and rinse the pellet 

with 500 μl of cold 70% ethanol. 

6. Resuspend the DNA in 20 μl of DEPCtreated 

water (#R0601). 

PCR Templates 

PCR products can serve as templates for 

in vitro transcription. The RNA polymerase 

promoter must be located upstream of the 

sequence to be transcribed. 

5.2. Avoiding RNase 

contamination 

Our reagents have been tested to ensure they 

are endo-, exodeoxyribonuclease, ribonuclease, 

and phosphatase free. However, an RNasefree 

working environment and RNase-free 

solutions are also critical factors for performing 

successful in vitro transcription. 

General recommendations to avoid RNase 

contamination: 

 

and reagents when working with RNA. 

 

reagents to avoid contact with skin, which 

is a source of RNases. Change gloves 

frequently. 

 

 

purification and handling with DEPC. Add 

DEPC to 0.1% (v/v) final concentration; 

incubate overnight at room temperature and 

autoclave. 

 

buffer solutions. Buffers containing Tris 

should be prepared by dissolving Tris base 

in DEPC-treated water. Solutions containing 

DTT or nucleotides should be prepared using 

DEPC-treated water and be passed through a 

0.2 μm filter for sterilization. 

 

use and all tubes tightly closed during the 

transcription reaction. 

5.3. In vitro transcription 

More than 10 μg of RNA transcript can be 

generated per 1 μg template DNA using the 

following protocol. The reaction can be scaled 

up or down. For high yield transcription, 

generating up to 200 μg RNA, use the 

TranscriptAid T7 High Yield Transcription Kit 

(#K0441). 

 

briefly. 

 

 

temperature. 

1. Prepare the following reaction mixture at 

room temperature in the order given: 

5X Transcription buffer 10 μl 

ATP/GTP/CTP/UTP Mix, 10 mM each 

10 μl 

(2 mM final 


Linearized template DNA 1 μg 

RiboLock RNase Inhibitor (#EO0381) 1.25 μl (50 u) 

T7/T3/SP6 RNA Polymerase 

(#EP0111, #EP0101, #EP0131) 

1.5 μl (30 u) 



2. 

3. Optional: To remove template DNA add 2 μl 

 

mix and incubate at 37°C for 15 min. 

4. 

extraction. 

Note 

RNA hydrolyzes if heated in the absence of a chelating 

agent. 

5.4. Synthesis of radiolabeled 

RNA probes of high specific 

activity 

1. Linearize template DNA with a restriction 

enzyme. Extract DNA with phenol/chloroform, 

then with chloroform/isoamyl alcohol, and 

precipitate with ethanol. Dissolve DNA in 

DEPC-treated Water (#R0601). 


336 

2. Combine the following reaction components 

at room temperature in the order given: 

5X Transcription buffer 4 μl 

3 NTP Mix, 10 mM each* (without labeled 

NTP) 

100 μM CTP (#R0451) 


1 μl 

(0.5 mM final 


2.4 μl 

(12 μM final 


[-32P]-CTP, ~30 TBq/mmol (800 Ci/mmol) 

1.85 MBq 

(50 μCi) 

Linear template DNA 0.2-1.0 μg 


T7/T3/SP6 RNA Polymerase (#EP0111, 

#EP0101, #EP0131) 

1 μl (20 u) 



3. 

4. Stop the reaction by cooling at -20°C. 

5. Determine the percentage of the label 

incorporated into RNA. 

Note 

* To prepare a mix of the three non-labeled NTPs (10 mM) 

each, combine 1 μl of all three NTPs, 100 mM, from the set 

(#R0481) with 7 μl of DEPC-treated Water (#R0601). Store 

the mix at -20°C for further use. 

 

3-5 x108 dpm/μg RNA. 

32P], [ 35S] or 

[ 3H]-ribonucleotides. Recommended amounts 

of radiolabeled nucleotides in a 20 μl of 

reaction mixture are as follows: 

Radiolabeled 

nucleotides 

Amounts for 20 μl of 


5’-[- 35 S]-UTP 11.1 MBq (300 μCi) 

5,6-[ 3 H]-UTP 0.925 MBq (25 μCi) 

5’-[- 32 P]-CTP 1.85 MBq (50 μCi) 

5.5. Purification of RNA 

transcripts 

Specific 

activity 

>37 TBq/mmol 

(1000 Ci/ mmol) 

1.1-2.2 TBq/mmol 

(30-60 Ci/mmol) 

~30 TBq/mmol 

(800 Ci/mmol) 

Template DNA 

Template DNA may interfere with downstream 

applications of the RNA transcript. Template 

 

directly after the transcription reaction. Add 2 u 

 

incubate at 37°C for 15 min, then add 2 μl of 

0.5 M EDTA, pH 8.0 (#R1021) and incubate at 

65°C for 10 min to stop the reaction. 

Proteins and nucleotides 

Phenol/chloroform extraction and ethanol 

precipitation of RNA transcripts is 

recommended. 

1. To a 50 μl reaction mixture, add 85 μl of 

DEPC-treated water (#R0601) and 15 μl 

of 3 M Sodium Acetate (#R1181). Mix 

thoroughly. 

2. Extract with an equal volume of 1:1 phenol/ 

chloroform mixture, and then twice with 

an equal volume of chloroform. Collect the 

aqueous phase and transfer to a new tube. 

3. Precipitate the RNA by adding 2 volumes 

 

least 30 min and collect the pellet by 

centrifugation. 

4. Remove the supernatant and rinse the pellet 

with 500 μl of cold 70% ethanol. 

5. Resuspend the RNA in 20 μl of DEPCtreated 

water (#R0601). 

6. Store the RNA at -20°C or -70°C. 

5.6. Evaluation of transcription 

reaction products 

Quantification by UV Light Absorbance 

The easiest way to determine RNA 

concentration is to measure UV absorbance at 

260 nm. Dilute an aliquot of the conventional 

transcription reaction 1:20 (1:300 for high yield 

transcription) to obtain an absorbance reading 

in the linear range of a spectrophotometer. 

For single-stranded RNA, when A260 = 1, RNA 

concentration is 40 μg/ml. The RNA yield can 

be calculated as follows: 

A260 x (dilution factor) x 40 = μg/ml RNA. 

Note 

Unincorporated nucleotides and template DNA in the 

mixture will interfere with quantification. Therefore, it 

is advisable to remove template and nucleotides from 

transcription mixture (see “Purification of RNA Transcripts” 

above). 

Sizing and Quantification by Gel 

Electrophoresis 

To evaluate transcript length, integrity and 

quantity, an aliquot of the transcription reaction 

should be run on a native or denaturing agarose 

gel or polyacrylamide gel along with the 

appropriate RNA ladder; e.g., RiboRuler High 

Range (#SM1821) or RiboRuler Low Range 

RNA Ladder (#SM1831). 

Transcript length Recommended gel 

>500 bases 1% agarose gel 

100-500 bases 

50-100 bases 


1.1 

Non-optimal 

reaction conditions 

1.3 

Insufficient yield 

of short transcript 

1.5 

Incorrect reaction 

preparation 

1 

Low yield or no 

RNA transcript 

Evaluate 

inhibition by 

template DNA 

1.2 

RNase 

contamination 

2 

Transcript is larger 

than expected 

Inhibition No inhibition 

1.4.1 

Reaction inhibitors 

in template DNA 

solution 

1.4 

DNA template of 

low purity or 

concentration 

1.4.2 

Insufficient 

amount of 

template 

Transcription problem 

2.1 

Incomplete 

cleavage of 

template DNA 

2.2 

3’-overhangs at 

DNA template 

ends 

2.3 

Aberrant 

migration of 

transcript 

3 

Transcript smearing 

on denaturing 

agarose gel 

3.1 

RNase 

contamination in 

template DNA 

3.2 

RNase 

contamination in 

environment 

4 

Truncated 

transcript 

4.1 

Termination 

signal in 

template DNA 

4.2 

GC-rich DNA 

template 


338 

Table 5.1. Troubleshooting guide for in vitro transcription. 


1. Low yield or no RNA transcript 


1.1. Non-optimal reaction conditions. 

A conventional in vitro transcription reaction using stand alone RNA polymerases should produce at least 

10 μg of RNA transcript from 1 μg of template. For higher RNA yields (up to 200 μg), the TranscriptAid T7 

High Yield Transcription Kit (#K0441) should be used. 

 

increase the yield of RNA by reducing the effect of reaction inhibition by pyrophosphates. 

1.2. RNase contamination. 

Working environment, DNA template, reagents or electrophoresis systems may be contaminated with 

RNases. 

 

 

 

Note 

RiboLock 

Do not use electrophoresis tanks which have been previously used for plasmid DNA miniprep analysis as they may be contaminated with 

RNases A or T1. 

1.3. Insufficient yield of short transcript. 

High yields of short transcripts (

Table 5.1. Troubleshooting guide for in vitro transcription. 


2. Transcript is larger than 

expected 

3. Transcript smearing 

on denaturing agarose gel 

4. Truncated transcript 

References 

1. Schenborn, E.T and Mierendorf, R.C., Nucl. Acids 

Res.,13, 6223-6236, 1985. 

2. Aziz, R.B. and Soreq, H., Nucl. Acids Res., 18, 

3418, 1990. 

2.1. Incomplete cleavage of template plasmid DNA. 

Even small amounts of undigested circular DNA can produce large amounts of long transcripts. Check the 

template for complete digestion and, if required, additionally digest with the appropriate restriction enzyme 

(see Troubleshooting Guide for DNA Digestion on p.229). For faster and more efficient plasmid cleavage, 

use FastDigest 

2.2. 3’-overhangs at DNA template ends. 

Avoid plasmid linearization with restriction enzymes that generate 3’-overhangs. Alternatively, blunt 

3’-overhangs with T4 DNA Polymerase (#EP0061) before use in transcription. See protocol on p.295. 

2.3. Aberrant migration of transcript. 

Due to secondary structures, RNA may run aberrantly on a native gel. On a denaturing gel, these transcripts 

normally migrate as single bands at the expected size. 

3.1. RNase contamination in template DNA. 

During preparation, plasmid DNA templates are often contaminated with RNases. This can affect the length 

and yield of synthesized RNA, and is seen as a smear below the expected transcript length 

Plasmid Miniprep Kit (#K0502), omit the RNase A from 

 

premixed in the purification buffers, perform phenol/chloroform extraction after plasmid DNA linearization, 

then ethanol precipitate the DNA and dissolve in DEPC-treated water (#R0601) 

(see Plasmid Templates on p.335). 

3.2. RNase contamination in working environment. 

 

 

*. 

 

they may be contaminated with RNases A or T1. 

* RiboLock 

4.1. RNA polymerase recognizes a termination signal in template DNA sequence. 

Try another RNA polymerase system or perform the transcription reaction at a lower temperature 

(e.g., 30°C). This may increase the length of transcript. However, RNA yield may be decreased 

at lower temperatures. 

4.2. GC-rich DNA template (or template with high secondary structure). 

For templates with secondary structure, incubating at 42°C or using a single-stranded binding (SSB) protein 

has been reported to improve yield and transcript length (2). 


340 


Thermo Scientific Molecular Labeling & 

Detection Products 


Products .............................................................................................................. 343 

Biotin DecaLabel DNA Labeling Kit.............................................................................343 

DecaLabel DNA Labeling Kit .......................................................................................344 

Biotin Chromogenic Detection Kit ..............................................................................345 



6.1. DNA/RNA end labeling ..............................................................................................349 

6.2. Random-primed labeling ...........................................................................................350 

6.3. DNA labeling by nick-translation ................................................................................350 

6.4. RNA labeling by in vitro transcription .........................................................................350 

6.5. Synthesis of labeled cDNA ........................................................................................350 

6.6. Southern Blotting ..................................................................................................... 351 


342 


Probe type Labeling strategy Radioactive 

DNA 

RNA 


Product group Radioactive 

Modified nucleotides 

dNTP 


Random primed labeling 

Nick-translation 

3’-end labeling 

Non- 

Radioactive 

Non- 

Radioactive 

Product Cat. # Page 

DecaLabel DNA Labeling Kit K0621/2 344 

Biotin DecaLabel DNA Labeling Kit K0651/2 343 

Klenow Fragment, exo – EP051/2/4 248 

DNA Polymerase I EP0041/2 246 

DNase I, RNase-free EN0521/3/5 255 

Klenow Fragment EP0051/2/4 247 

T4 DNA Polymerase EP0061/2 249 

T7 DNA Polymerase EP0081 250 

Terminal Deoxynucleotidyl Transferase (TdT) EP0161/2 251 

5’-end labeling T4 Polynucleotide Kinase (T4 PNK) EK0031/2 243 

In vitro transcription 

TranscriptAid T7 High Yield Transcription Kit K0441 328 


SP6 RNA Polymerase EP0131/3 331 


3’-end labeling T4 RNA Ligase EL0021 240 

3’-end labeling Terminal Deoxynucleotidyl Transferase (TdT) EP0161/2 251 

5’-end labeling T4 Polynucleotide Kinase (T4 PNK) EK0031/2 243 

Product Cat. # Page 

Biotin-11-dUTP R0081 414 

Fluorescein-12-dUTP R0101 413 

Aminoallyl-dUTP R0091/1101 414 

Aminoallyl-UTP R1091 414 

dNTP Set R0181/2/6 408 




NTP NTP Set R0481 411 

Oligo(dT) 18 Primer SO131/2 417 

Primers 

Random Hexamer Primer SO142 417 

Exo-Resistant Random Primer SO181 417 

Water 

 

 

 

 



R0581/2 

R0601/3 

420 

420 

Detection kit Biotin Chromogenic Detection Kit K0661/2 345 

Chromogenic substrates 

 

 

BCIP-T 

NBT 

R0821/2 

R0841/2 

426 

426

Products 

Biotin DecaLabel DNA Labeling Kit 




Biotin Chromogenic Detection Kit p.345 

BCIP-T p.426 

NBT p.426 

GeneJET Genomic DNA Purification Kit p.310 



GeneJET 

 

 

GeneJET Plant Genomic DNA 


Agarase p.319 

5’ 

3’ 

5’ 

3’ 

5’ 

3’ 

denaturation and 

annealing with 

random primers 

Principle of DNA labeling by the random primed 

method. 

* [-32P]-dNTP, [-33P]-dNTP, biotin-dUTP, 

fluorescein-, aminoallyl- or DIG-dUTP can be u sed. 

5’ 3’ 

DNA template 

random primer 

labeled probe 

References 

1. Feinberg, A.P., Vogelstein, B., A technique for 

radiolabeling DNA restriction endonuclease fragments to 

high specific activity, Biochem., 132, 6-13, 1983. 


radiolabeling DNA restriction endonuclease fragments to 

high specific activity, Addendum, Biochem., 137, 

266-267, 1984. 

3’ 

5’ 

3’ 

5’ 

Klenow Fragment, exo 

3’ 

– 

dNTP 

labeled dNTP* 

denaturation 

5’ 

Description 

The Thermo Scientific Biotin DecaLabel DNA 

Labeling Kit is an advanced system for efficient 

synthesis of biotin-labeled DNA probes, based 

on an improved random-primed labeling method 

originally developed by Feinberg and Vogelstein 

(1, 2). The primary improvement over the 

traditional random-primed method involves the 

use of random decamers instead of hexamers 

to ensure more efficient annealing with DNA at 

37°C. Klenow Fragment, exo – is also included in 

the kit; this genetically engineered enzyme has 

no detectable exonuclease activity. Therefore, 

the enzyme does not degrade the labeled probe 

during the reaction, which results in a high 

labeling yield even with low amounts of template. 

As a result, DNA fragments of any length can 

be uniformly labeled. Biotin-labeled DNA can be 

detected with the Biotin Chromogenic Detection 

Kit (#K0661) or with conventional biotin-avidin or 

biotin-streptavidin detection systems. 

Features 

Non-radioactive labeling of DNA. 

Efficient priming of labeling reactions with 

random decamers. 

High yields with Klenow Fragment, exo – : no 

degradation of the labeled probe during reaction. 

Application 

Generation of biotin-labeled DNA probes 

for a variety of non-radioactive hybridization 

experiments, including Southern and Northern 

blots, colony/plaque hybridizations, dot/slot blots 

and in situ hybridizations. 

Principle 

Random decamers are annealed to a denatured 

template DNA molecule and new strands are 

synthesized by Klenow Fragment, exo – in the 

presence of biotin-dUTP. During this reaction, 

the biotinylated nucleotides are incorporated 

into the newly synthesized complementary DNA 

strand. 


– 

 

 

 

 

 

 

300pg 100pg 30pg 10pg 3pg 1pg 0.3pg 0.1pg 0.03pg 0.01pg 

Dot-blot hybridization with a biotin-labeled probe. 

Lambda DNA/HindIII was biotin-labeled with the Biotin DecaLabel DNA Labeling 

Kit and used as a hybridization probe in a dot-blot of homologous DNA on 

a nylon membrane. The blot was developed using the Biotin Chromogenic 

Detection Kit (#K0661). 


» 6.6. Southern Blotting p.351 


344 

DecaLabel DNA Labeling Kit 







GeneJET 

 

 

GeneJET Plant Genomic DNA 



Silica Bead DNA Gel Extraction Kit p.318 

Agarase p.319 




Description 

The Thermo Scientific DecaLabel DNA Labeling 

Kit is an advanced system for fast synthesis 

of radiolabeled DNA probes of high specific 

activity. The kit is based on the improved 

random-primed method developed by Feinberg 

and Vogelstein (1, 2). 

The primary improvement over traditional 

random-primer kits involves the use of random 

decamers instead of hexamers to ensure 

more efficient annealing with DNA at 37°C. 

Klenow Fragment, exo – is also included in the 

kit; this genetically engineered enzyme has 

no detectable exonuclease activity. Therefore, 

the enzyme does not degrade the labeled 

probe during the reaction, which results in 

a high labeling yield even with low amounts 

of template. DNA fragments of any length 

can be uniformly labeled. Two labeling mixes 

are provided for flexibility in using either 

radiolabeled dATP or dCTP. 

Features 

High specific radioactivity of probes – 

>1x10 9 dpm/μg DNA. 

Fast – 5 minutes at 37°C. 

High yields with Klenow Fragment, exo – : 

no degradation of the labeled probe during 

reaction. 

Flexible – suitable for either radiolabeled 

dATP or dCTP. 

Specific radioactivity (x10 9 dpm/μg DNA) 

5 

4 

3 

2 

1 

0 

0 

5 

10 

Time (min) 

Application 

Generation of radiolabeled DNA probes for use 

in a variety of hybridization experiments: 

Southern and Northern blots, colony/plaque 

hybridizations, dot/slot blots and in situ 

hybridizations. 

Principle 

Random decamers are annealed to a denatured 

template DNA, and radiolabeled dNTPs are then 

incorporated into new DNA strands by Klenow 

Fragment, exo – . 


– 

 

 

 

 

 

 

 

12.5 ng 

25 ng 

50 ng 

100 ng 

200 ng 

Effect of reaction time and amount of template on the specific radioactivity of a DNA probe. 

Varying amounts of DNA/HindIII fragments were labeled with 50 μCi [- 33 P]-dCTP (2500 Ci/mmol) 

according to the protocol supplied with the kit. 

15 

20 

References 


radiolabeling DNA restriction endonuclease fragments 

to high specific activity, Biochem., 132, 6-13, 1983. 


radiolabeling DNA restriction endonuclease fragments 

to high specific activity, Addendum, Biochem., 

137, 266-267, 1984.

Biotin Chromogenic Detection Kit 




Biotin DecaLabel DNA Labeling Kit p.343 

Biotin-11-dUTP p.414 

NBT p.426 

BCIP-T p.426 

Color 

precipitate 

Chromogenic detection of 

a biotin-labeled probe. 


Streptavidin 

Biotin 

Probe 

Description 

The Thermo Scientific Biotin Chromogenic 

Detection Kit is a convenient tool for 

chromogenic detection of biotinylated nucleic 

acid probes. The kit is optimized to reproducibly 

provide high sensitivity with low background in 

applications such as Southern, Northern, dot 

and slot blotting, as well as screening of viral 

plaques and bacterial colonies. Biotinylation 

of DNA and RNA probes is widely used as a 

safe and convenient alternative to radioactive 

labeling. Biotin can be incorporated into nucleic 

acids using various enzymatic or non-enzymatic 

methods including the Biotin DecaLabel DNA 

Labeling Kit (#K0651). Biotinylated probes are 

detected with streptavidin coupled to alkaline 

phosphatase (AP). Streptavidin-AP conjugates 

bind specifically and irreversibly to the biotinlabeled 

probes. The probes are then visualized 

using a chromogenic substrate for alkaline 

phosphatase -BCIP/NBT, which produces a 

blue-purple precipitate. Therefore, visualization 

does not require X-ray film or other specific 

equipment. 

The Biotin Chromogenic Detection Kit includes 

 

which are specifically designed to minimize 

the background often associated with biotin 

detection in conventional systems. 

Target 

300pg 100pg 30pg 10pg 3pg 1pg 0.3pg 0.1pg 0.03pg 0.01pg 

Dot-blot hybridization with a biotin-labeled probe. 

Lambda DNA/HindIII was biotin-labeled with the Biotin DecaLabel DNA 

Labeling Kit (#K0651) and used as a hybridization probe in a dot-blot of the 

homologous DNA on the nylon membrane. The blot was developed with the Biotin 

Chromogenic Detection Kit. 

Features 

Highly sensitive detection – 0.03 pg of 

homologous DNA in dot blot hybridization 

with a biotin-labeled probe. 

Low background due to optimized washing 

and blocking procedures. 

Visualization of non-radioactive detection 

does not require X-ray film or other 

specific equipment. 

Fast and convenient – ready-to-use 

components. 

Applications 

Southern blots. 

Northern blots. 

Dot/slot blots. 

Plaque or colony screening. 


 

 

 

 

 

 




346 


DNA/RNA 5’-end labeling Cat. # Size Supplied with Applications Page 

T4 Polynucleotide Kinase (T4 PNK), 

10 u/μl 


EK0031 500 u 

EK0032 2500 u 




10X Reaction Buffer A 2 ml 




» 6.1.1. DNA 5’-end labeling by T4 PNK in the exchange reaction 

» 6.1.2. DNA/RNA 5’-end labeling by T4 PNK in the forward reaction 

» 6.1.3. Radiolabeling of RNA Ladders by T4 PNK 

Radioactive labeling of 5’-termini of 

single and double-stranded DNA and 

RNA, such as: 

– DNA and RNA molecular weight 

markers, 

– sequencing primers, 

– PCR primers, 

– DNA and RNA oligonucleotides or 

linkers, 

– hybridization probes, 

– probes for transcript mapping. 

DNA 3’-end labeling by fill-in Cat. # Size Supplied with Applications Page 

Klenow Fragment, 10 u/μl 

EP0051 

EP0052 

300 u 

1500 u 



1 ml 

5x1 ml 

247 

Klenow Fragment, 2 u/μl EP0054 LC, 300 u 10X Reaction Buffer 1 ml DNA 3’-end labeling by fill-in of 

T4 DNA Polymerase, 5 u/μl 

EP0061 

EP0062 

100 u 

500 u 



0.35 ml 

2x1 ml 

5’-overhangs. 

249 

T7 DNA Polymerase, 10 u/μl EP0081 300 u 10X Reaction Buffer 0.4 ml 250 

243 

p.349 

p.349 

p.349 


» 6.1.4. DNA 3’-end labeling by fill-in of 5’-overhangs p.349 

3’-end labeling by tailing Cat. # Size Supplied with Applications Page 

Terminal Deoxynucleotidyl 

Transferase (TdT), 20 u/μl 

EP0161 500 u 5X Reaction Buffer 0.4 ml Oligodeoxyribonucleotide and DNA 

EP0162 2500 u 5X Reaction Buffer 2x1 ml 3’-termini labeling by tailing. 


» 6.1.5. DNA and oligonucleotide 3’-end labeling by tailing p.349 

3’-end labeling by ligation Cat. # Size Supplied with Applications Page 

T4 RNA Ligase, 10 u/μl EL0021 1000 u 


 

1 mg/ml BSA Solution 0.2 ml 

RNA and double-stranded 

DNA 3’-end labeling by ligation. 


» 6.1.6. RNA 3’-end labeling by ligation p.350 

251 

240

Random-primed labeling Cat. # Size Supplied with Applications Page 

Klenow Fragment, 10 u/μl 

EP0051 300 u 10X Reaction Buffer 1 ml 

EP0052 1500 u 10X Reaction Buffer 5x1 ml 

Klenow Fragment, 2 u/μl EP0054 LC, 300 u 10X Reaction Buffer 1 ml 

Klenow Fragment, exo – , 5 u/μl 



Random-primed DNA labeling. 


» 6.2. Random-primed labeling p.350 

Chromogenic substrates Cat. # Size Applications Page 

BCIP-T 

NBT 

R0821 1 g 

R0822 5 g 

R0841 1 g 

R0842 5 g 

Detection of alkaline phosphatase on Southern, Northern, 

dot/slot blots, plaque or colony screening. 



Nick-translation Cat. # Size Supplied with Applications Page 

DNA Polymerase I, 10 u/μl 


DNase I, RNase-free, 50 u/μl EN0523 HC,1000 u 

DNase I, RNase-free, 1 u/μl 

2) 



EN0525 1000 u 

2 1 ml 

 

2 1 ml 

2 1 ml 

2 1 ml 

 

DNA labeling by 

nick-translation. 


» 6.3. DNA labeling by nick-translation p.350 

247 

248 

426 

426 

246 

255 

255 


348 

RNA labeling 

by in vitro transcription 

T7 RNA Polymerase, 20 u/μl 

T7 RNA Polymerase, >100 u/μl EP0113 

Nucleotides for labeling Cat. # Size Applications Page 

Biotin-11-dUTP, R0081 50 nmol (50 μl) 

Fluorescein-12-dUTP, R0101 25 nmol (25 μl) 413 

Aminoallyl-dUTP, R0091 1 μmol (100 μl) 

Aminoallyl-dUTP, R1101 2.5 μmol (50 μl) 

Aminoallyl-UTP, R1091 2.5 μmol (50 μl) 

dNTP Set, 


Cat. # Size Supplied with Applications Page 

EP0111 5000 u 5X Transcription Buffer 1.25 ml 

EP0112 5x5000 u 

HC, 

25000 u 


SP6 RNA Polymerase, 20 u/μl EP0131 2000 u 5X Transcription Buffer 0.5 ml 

SP6 RNA Polymerase, >100 u/μl EP0133 HC, 5000 u 5X Transcription Buffer 1.25 ml 

T3 RNA Polymerase, 20 u/μl 

T3 RNA Polymerase, >100 u/μl EP0103 

TranscriptAid T7 High Yield 

Transcription Kit 

EP0101 2000 u 5X Transcription Buffer 0.5 ml 

EP0102 10000 u 

HC, 

10000 u 


K0441 50 reactions – 

R0181 (4x25 μmol) 4x0.25 ml 

R0182 (4x100 μmol) 4x1 ml 

R0186 (4x500 μmol) 4x5 ml 

Synthesis of labeled RNA probes for: 

– blotting, 

– in situ hybridization, 

– microarray hybridization. 

Generation of non-radioactively 

labeled RNA probes. 


» 5.4. Synthesis of radiolabeled RNA probes of high specific activity p.335 

All types of enzymatic labeling. 

330 

331 

331 

328 

414 

414 

408


6.1. DNA/RNA end labeling 

6.1.1. DNA 5’-end labeling by T4 PNK in 

the exchange reaction 

All types of DNA ends can be successfully 

labeled with T4 Polynucleotide Kinase. 

However, the labeling efficiency is greatest for 

the 5’-protruding DNA ends, lower for blunt 

ends, and is the lowest for 5’-recessed DNA 

ends. 

This protocol is recommended for radiolabeling 

of DNA markers and ladders (ready-to-use 

versions with the loading dye pre-mixed are not 

suitable for labeling). 


1-20 pmol of 

Linear DNA 

5’-termini 

10X reaction buffer B for T4 PNK 2 μl 

[-32P or -33P]-ATP 40 pmol 

24% (w/v) PEG 6000 solution 4 μl 





3. 

incubate at 75°C for 10 min. 

4. Separate labeled DNA from unincorporated 

label by gel filtration on Sephadex G-50. 

Note 

-32P or -33P]-ATP is used, 

 

nuclease-free (#R0581). 

 

see Appendix 

p.465 or www.thermoscientific.com/reviewer. 

6.1.2. DNA/RNA 5’-end labeling by 

T4 PNK in the forward reaction 


Dephospharylated DNA or 


10X reaction buffer A for T4 

Polynucleotide Kinase 

1-20 pmol of 5’-termini 

10-50 pmol 

2 μl 

[-32P or -33P]-ATP 20 pmol 





3. 

incubate at 75°C for 10 min. 

4. Separate labeled DNA from unincorporated 


Note 

-32P or -33P]-ATP is used, 

 

nuclease-free (#R0581). 

 

see Appendix 

p.465 or www.thermoscientific.com/reviewer. 

6.1.3. Radiolabeling of RNA Ladders 

by T4 PNK 

The ready-to-use versions of the RiboRuler 

RNA ladders can not be radiolabeled with T4 

PNK. 

For efficient labeling of RNA ladders it is 

recommended to remove the 5’-phosphate 

groups from the RNA and then phosphorylate 

in the forward reaction using T4 Polynucleotide 

Kinase. 

I. Dephosphorylation. 


RiboRuler Low Range RNA Ladder or 

RiboRuler High Range RNA Ladder 

8 μl 


10X reaction buffer for alkaline phosphatase 2 μl 


(#EF0651) 

2 μl (2 u) 




3. Remove the enzymes from the mixture with 

20 μl of Tris-saturated ( pH 8.0) phenol 

chloroform mixture. Save the upper aqueous 

phase and extract it twice with 20 μl of 

chloroform. 

4. 

Sodium Acetate Solution (#R1181) and 55 μl 

of 96% ethanol. Store at -20°C for 15-30 

min. Centrifuge the mixture at 10000- 

15000 rpm and 4°C for 20 min. 

5. Rinse the pellet with 20 μl of cold 75% 

ethanol. Centrifuge 10 min at 10000- 

15000 rpm, 4°C for 10 min. 

6. Discard the supernatant and dissolve the 

air-dried pellet in 10 μl of DEPC-treated 

 

II. Labeling 


Dephosphorylated RNA Ladder 1 μl 2.5 μl 

[-32P]-ATP (5000 Ci/mmol,10 μCi/μl)* 5 μl 

(10 pmol) 


10X buffer A for forward reaction 

(supplied with T4 PNK) 

1 μl 




* If [- 32 P]-ATP with a high specific activity (higher than 5000 

Ci/mmol) is used, the label can be diluted with cold ATP 

(#R0441). Total 


3. 

EDTA, pH 8.0 (#R1021) and extract the 

mixture with an equal volume of chloroform. 

4. Determine the efficiency of label 

incorporation. 

6.1.4. DNA 3’-end labeling by fill-in of 

5’-overhangs 


Linear DNA 0.1-4 μg 

10X reaction buffer for Klenow Fragment 2 μl 

[-32P]-dNTP, ~15-30 TBq/mmol (400-800 Ci/mmol) 

or 

[-32 

(20 μCi) 2.96 

P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol) 

3 dNTP Mix, 2 mM each 

(without a labeled dNTP) 

2.5 μl 

 

final concentration) 

Klenow Fragment (#EP0051) 0.1 μl (1 u) 





10 min. 

Note 

This protocol is suitable for labeling of the following Thermo 

Scientific DNA markers, composed of DNA fragments with 

5’-overhangs: 

Lambda DNA EcoRI Marker 

Lambda DNA HindIII Marker 

Lambda DNA EcoRI/HindIII Marker 

The modified version of this protocol can be used for nonradioactive 

labeling of DNA markers. Substitute a part of 

dTTP with a modified nucleotide (e.g., Biotin-11-dUTP or 

Fluorescein-12-dUTP) at a molar ratio of 1:2. 

6.1.5. DNA and oligonucleotide 3’-end 

labeling by tailing 


5X reaction buffer for TDT 10 μl 

Linear DNA 10 pmol 

[-32P]-ddATP, ~110 TBq/mmol (3000 Ci/mmol) 

 

(50 μCi) 

Terminal Deoxynucleotidyl Transferase (#EP0161) 2 μl (40 u) 





 

(#R1021). 

Note 

The efficiency of the reaction depends upon the type of 

3’-OH termini of the DNA fragments. 3’-protruding ends are 

labeled with higher efficiency than recessed or blunt ends. 

References 

 

 


 

 

York, 3.10.2-3.10.5, 1994-2004. 


350 

6.1.6. RNA 3’-end labeling by ligation 

1. Prepare the following reaction mixture in a 

RNase-free microfuge tube: 

10X ligation buffer for T4 RNA Ligase 2 μl 

10 mM ATP 1 μl 

RNA 50-100 pmol 

[ 32P]-pCp 50-100 pmol 

(equimolar amount) 

T4 RNA Ligase (#EL0021) 1 μl (10 u) 



2. Incubate at 4°C for 10-12 hours (overnight). 

3. Separate labeled RNA from unincorporated 


6.2. Random-primed labeling 

6.2.1. Radioactive random-primed 

DNA labeling 


DNA template 10 μl (100 ng) 

10X reaction buffer for Klenow Fragment, exo – 5 μl 

6.0 A260units/ml (100 μM) Random Hexamer 


12.5 μl 



2. Incubate the mixture in a boiling water bath 

for 5-10 min and then chill on ice. 

3. Add: 

3 dNTP Mix, 0.33 mM each (without a 

labeled dNTP) 

[-32P]-dNTP, ~110 TBq/mmol (3000 Ci/ 

mmol) 


 


 

Klenow Fragment, exo – (#EP0421) 1 μl (5 u) 



4. Incubate the reaction mixture at 37°C for 

10 min. 

5. 

at 37°C for 5 min. 

6. 

stop the reaction. 

7. Remove 1 μl of the reaction mixture and 

determine the percentage of incorporated 

label. 

8. Purify by using Sephadex G-50 or Bio-Gel 

P-60. 

6.2.2. Non-radioactive randomprimed 

DNA labeling 


DNA template 

10 μl (100 – 

1 μg) 

10X reaction buffer for Klenow Fragment, exo – 5 μl 

6.0 A260units/ml (100 μM) Random Hexamer 


12.5 μl 



2. Incubate the mixture in a boiling water bath 

for 5-10 min and then chill on ice. 

3. Add: 

3 dNTP Mix, 1 mM each 

(without the dTTP) 

 


dTTP, 1 mM* 

 

conc.) 

Biotin-11-dUTP**, 1 mM (#R0081) 1.75 μl 

Klenow Fragment, exo – (#EP0421) 1 μl (5 u) 


* 

ATP solution (#R0441) and 99 μl of water, nuclease-free. 

** Fluorescein-12-dUTP (#R0101), DIG-dUTP or AminoallyldUTP 

(#R0091) can also be used with the same protocol. 

4. Incubate the reaction mixture at 37°C for 

1 h. 

5. 

stop the reaction. 

6. Remove 1 μl of the reaction mixture and 

determine the percentage of incorporated 

label. 

7. Optional: purify by using Sephadex G-50 or 

Bio-Gel P-60. 

6.3. DNA labeling by nicktranslation 

6.3.1. Radioactive DNA labeling by 

nick-translation 

1. 

10X reaction buffer for DNA Polymerase I 2.5 μl 

Mixture of 3 dNTPs, 1mM* (without the labeled dNTP) 1.25 μl 

[-32P]-dNTP ~110 TBq/mmol (3000 Ci/mmol) 

DNase I, RNase-free (#EN0521) 

freshly diluted to 0.002 u/μl** 

DNA Polymerase I, E.coli (#EP0041) 

 

(50-100 μCi) 

1 μl 

0.5-1.5 μl 

(5-15 u) 

Template DNA 0.25 μg 



2. Immediately incubate at 15°C for 

15-60 min. 

3. 

EDTA, pH 8.0 (#R1021). 

4. Take an aliquot (1 μl) to determine the 

efficiency of the label incorporation. A 

specific activity of at least 10 8 cpm/μg DNA 

is expected. 

5. If needed, the labeled DNA may be 

separated from the unincorporated 

radioactive precursors on Sephadex G-50 or 

Bio-Gel P-60 column. 

Note 

* 

of each), mix 1 μl aliquots of stock solutions of each dNTP 

 

(#R0581). These dNTP mixes can be stored at -20°C for 

further use. 

** DNase I, RNase-free (#EN0521) can be diluted with 1X 

reaction buffer for DNA Polymerase I (#EP0041). 

 

can be prepared using two radioactively labeled dNTPs 

simultaneously. In this case, the composition of the 

unlabeled dNTP mix should be adjusted accordingly. 

6.3.2. Non-radioactive DNA labeling 

by nick-translation 

The protocol 6.3.1. can be used for nonradioactive 

labeling by Nick-translation using 

biotin-11-dUTP, fluorescein-12-dUTP, DIG-dUTP 

or aminoallyl-dUTP: 

 

a molar ratio of 1:3-1:5, 

 

6.4. RNA labeling by in vitro 

transcription 

6.4.1. Synthesis of radiolabeled RNA 

probes of high specific activity 

1. Linearize template DNA with a restriction 

enzyme. Extract DNA with phenol/ 

chloroform, then with chloroform/isoamyl 

alcohol, and precipitate with ethanol. 

 

(#R0601). 

2. Combine the following reaction components 

at room temperature in the order given: 


5X transcription buffer 4 μl 

3 NTP Mix, 10 mM each* (without labeled NTP) 

 


 

100 μM CTP (#R0451) 


[-32P]-CTP, ~30 TBq/mmol (800 Ci/mmol) 

Linearized template DNA 0.2-1.0 μg 


T7 RNA Polymerase (#EP0111) or 

SP6 RNA Polymerase (#EP0131) or 

1 μl (20 u) 

T3 RNA Polymerase (#EP0101) 


3. Incubate at 37°C for 2 hours. 

4. Stop the reaction by cooling to -20°C. 

5. Determine the percentage of label 

incorporated into RNA. 

Note 

* 

 

 

the mix at -20°C for further use. 

8 dpm/μg. 

32P], [ 35S] or 

[ 3H]-ribonucleotides. Recommended amounts of 

radiolabeled nucleotides in a 20 μl of reaction mixture are 

as follows: 

Radiolabeled 

nucleoside 

Amounts for 20 μl of 


5’-[- 32 P]-CTP 

5’-[- 35 S]-UTP 

5,6-[ 3 H]-UTP 

Specific 

activity 

~30 TBq/mmol 

(800 Ci/mmol) 

>37 TBq/mmol 

(1000 Ci/mmol) 

1.1-2.2 TBq/mmol 

(30-60 Ci/mmol) 

6.5. Synthesis of labeled cDNA 

6.5.1. Synthesis of cDNA probes with 

high specific radioactivity 

This protocol is provided for first strand cDNA 

synthesis using RevertAid

iefly centrifuge all components after thawing, 

keep on ice. 

1. Add into a sterile, nuclease-free tube on ice 

in the order given: 

Template Total RNA or up to 5 μg 

RNA 

Poly(A) RNA or up to 500 ng 

Specific RNA or up to 500 ng 

Primers Oligo(dT) 18 (#SO131) or 0.5 μg (100 pmol) 

Random Hexamer (#SO141) or 0.2 μg (100 pmol) 

Gene-specific 15-20 pmol 

DEPC-treated Water (#R0601) to 8.5 μl 

Total volume 8.5 μl 

2. 

Optional. Incubate at 65°C for 5 min, chill 

on ice and briefly centrifuge. Perform this 

step if RNA template is GC-rich or is known 

to contain secondary structures. 

3. Place the tube with the primer/template mix 

on ice and add the following components in 

the indicated order: 

5X reaction buffer 4 μl 


dGTP, dCTP, dTTP mix, 10 mM each 1 μl 

0.1 mM dATP 4 μl 

[-32P]-dATP, 3000 Ci/mmol 1 μl 

RevertAid H Minus Reverse 

Transcriptase (Thermo Scientific) 

1 μl (200 u) 

Total volume: 20 μl 

4. 

5. If oligo(dT) 18 primer or a gene-specific 

primer is used, incubate at 42°C for 60 min. 

If random hexamers are used, incubate 

at 25°C for 10 min followed by 60 min at 

42°C. For transcription of GC-rich RNA the 

reaction temperature can be increased to 

45°C. 

6. 

EDTA, pH 8.0 (#R1021). 

Optional. Hydrolyze the RNA by the 

 

NaOH and incubation at 70°C for 30 min. 

7. Remove unincorporated dNTPs by 

chromatography on a Sephadex G-50 

column.Expect specific radioactivity of >10 7 

dpm/μg. 

Note 

To achieve higher specific activities (over 10 8 dpm/μg), use 

up to 100 μCi of [- 32 P]-dATP in the labeling mixture. To 

keep the total reaction volume at 20 μl, vacuum-dry 10 μl of 

[- 32 P]-dATP (10 mCi/ml) to 1 μl in a separate tube. 

6.5.2. Synthesis of non-radioactively 

labeled cDNA 

The protocol 6.5.1. can be used for synthesis 

of non-radioactive labeled cDNA using biotin- 

11-dUTP, fluorescein-12-dUTP, DIG-dUTP or 

aminoallyl-dUTP: 

– unlabeled dTTP is subsituted for 

labeled-dUTP at a molar ratio of 1:3-1:4, 

– reaction time is prolonged to 2-6 hours. 

6.6. Southern Blotting 

Required solutions (for preparation, 

see Appendix, p.467) 

1. Denaturation Solution: 

NaOH. 

2. Neutralization Solution: 

 

3. 20X SSC, pH 7.0 (blotting buffer): 

 

4. 100X Denhardt’s solution: 2% (w/v) BSA, 2% 

(w/v) Ficoll , 2% (w/v) PVP (polyvinylpyrrolidone). 

5. Pre-hybridization Solution: 6X SSC, 5X 

Denhardt’s solution, 50% formamide, 0.5% SDS. 


Load genomic DNA probes along with a 

DNA marker on a 0.7% agarose gel (20 cm 

length). Run for 18 hours at 3 V/cm in 1X 

TAE buffer. 

Southern Blotting 

1. Rinse the gel in deionized water, add 

Denaturation Solution and gently shake at 

room temperature for 30 min. Rinse the gel 

in deionized water and add Neutralization 

Solution. Shake at room temperature for 

15 min. Repeat neutralization procedure. 

 

a platform and cover it with a sheet of 

 

20X SSC buffer. 

2. Place the gel upside down on the filter and 

avoid trapping air bubbles beneath it. 

3. Cut a sheet of membrane to match the size 

of the gel and place it on the top of the 

gel. Avoid trapping air bubbles beneath the 

membrane. 

4. 

paper to the size of the gel, wet with 

blotting buffer and place on the top of the 

membrane. 

5. Place a stack of absorbent paper towels on 

 

the top of the paper towels and put a 0.5 kg 

weight on top. 

6. Allow upward capillary transfer of DNA at 

room temperature for 18 hours. 

7. 

to remove any residual agarose, dry at 

room temperature and fix for 2 min under 

UV-light. 

Generation of Labeled Probes 

The labeled probes are prepared using Biotin 

DecaLabel DNA Labeling Kit (#K0651), 

DecaLabel DNA Labeling Kit (#K0621) or using 

the protocol on p.350 for random-primed 

labeling. 

Hybridization 

1. Prepare 30 ml of the pre-hybridization 

solution. 

2. Denature sonicated salmon sperm DNA 

solution (10 mg/ml) by heating at 100°C 

for 5 min. Chill on ice and add to the 

pre-hybridization solution with a final 

concentration of 50-100 μg/ml. 

3. Place the membrane into the hybridization 

container, add pre-hybridization solution with 

the denatured salmon sperm DNA 

(0.2 ml/cm2 of membrane) and pre-hybridize 

at 42°C with shaking for 2 hours. 

4. Prepare the hybridization solution: 

 

at 100°C for 5 min and chill immediately 

on ice. 

Add the following amounts of the probe 

mixture to the pre-hybridization solution: 

10 ng/ml (1/5 of probe mix) if specific 

activity is 108 dpm/μg, 

 

activity is 109 dpm/μg, 

 

probes. 

5. Discard the pre-hybridization solution (from 

step 3) and add the prepared hybridization 

solution to the hybridization container (60 μl/ 

cm2 ). Incubate for at least 12 hours at 42°C. 

6. Carry out the following washes of the 

membrane: 

 

temperature for 10 min, 

 

for 10 min (for high stringency). 

7. 

 

Autoradiography 

and 

expose to a phosphoimager or a film with an 

intensifying screen. 

Upward capillary transfer of DNA from 

agarose gels. 


352 



DNA Electrophoresis Products 

Selection Guide ................................................................................................................354 

Products ............................................................................................................................358 

GeneRuler and O’GeneRuler DNA Ladders (10-20000 bp) .........................................358 

GeneRuler 1 kb DNA Ladder ......................................................................................359 

GeneRuler 1 kb Plus DNA Ladder ..............................................................................359 

GeneRuler DNA Ladder Mix .......................................................................................359 

GeneRuler 100 bp DNA Ladder..................................................................................359 

GeneRuler 100 bp Plus DNA Ladder ..........................................................................359 

GeneRuler 50 bp DNA Ladder ...................................................................................359 

GeneRuler Ultra Low Range DNA Ladder ....................................................................359 

GeneRuler Low Range DNA Ladder ............................................................................359 

GeneRuler High Range DNA Ladder ...........................................................................359 

GeneRuler Express DNA Ladder ................................................................................359 

MassRuler DNA Ladders, ready-to-use (80-10000 bp) .............................................362 

MassRuler Low Range DNA Ladder ...........................................................................362 

MassRuler Express LR Forward DNA Ladder ..............................................................362 

MassRuler Express LR Reverse DNA Ladder ..............................................................362 

MassRuler High Range DNA Ladder ...........................................................................362 

MassRuler Express HR Forward DNA Ladder .............................................................362 

MassRuler Express HR Reverse DNA Ladder ..............................................................362 

MassRuler DNA Ladder Mix .......................................................................................362 

MassRuler Express Forward DNA Ladder Mix.............................................................362 

MassRuler Express Reverse DNA Ladder Mix .............................................................362 

FastRuler DNA Ladders, ready-to-use (10-10000 bp)................................................364 

FastRuler Ultra Low Range DNA Ladder .....................................................................364 

FastRuler Low Range DNA Ladder .............................................................................364 

FastRuler Middle Range DNA Ladder .........................................................................364 

FastRuler High Range DNA Ladder ............................................................................364 

ZipRuler Express DNA Ladder Set, ready-to-use (100-20000 bp) ............................365 

O’RangeRuler DNA Ladders, ready-to-use (10-6000 bp) ..........................................366 

O’RangeRuler 5 bp DNA Ladder ................................................................................366 

O’RangeRuler 10 bp DNA Ladder...............................................................................366 

O’RangeRuler 20 bp DNA Ladder ..............................................................................366 

O’RangeRuler 50 bp DNA Ladder ..............................................................................366 

O’RangeRuler 100 bp DNA Ladder ............................................................................366 



O’RangeRuler 100+500 bp DNA Ladder ...................................................................366 

Conventional DNA Markers (8-23130 bp) ....................................................................368 

Lambda DNA/EcoRI Marker, 1 ...................................................................................368 

Lambda DNA/HindIII Marker, 2 ..................................................................................368 

Lambda DNA/EcoRI+HindIII Marker, 3 .......................................................................368 

pBR322 DNA/BsuRI (HaeIII) Marker, 5 .......................................................................368 

X174 DNA /BsuRI (HaeIII) Marker, 9 ........................................................................368 

pUC19 DNA/MspI (HpaII) Marker, 23 .........................................................................368 

NoLimits Individual DNA Fragments and Custom DNA Ladders .............................. 370 

TopVision Agarose ....................................................................................................... 371 

Electrophoresis Buffers .............................................................................................. 371 

Loading Dyes ................................................................................................................ 372 


7.1. General recommendations for DNA electrophoresis .................................................... 374 

7.2. Preparation of agarose gels for DNA electrophoresis .................................................. 374 

7.3. Preparation of gels for PAGE ..................................................................................... 375 

7.4. Preparation of DNA samples for electrophoresis ......................................................... 376 

7.5. Preparation of DNA ladders/markers for electrophoresis ............................................ 376 

7.6. Labeling of DNA ladders/markers .............................................................................. 376 

7.7. Separation of Express DNA ladders in different electrophoresis conditions .................. 377 

Troubleshooting Guide ................................................................................................... 378 

Find out more about 

DNA Electrophoresis 

products online: 

www.thermoscientific.com/ladders 


354 


Product lines of DNA ladders and markers 

DNA ladder/marker 

group 

NoLimits Custom 

DNA Ladders 

GeneRuler and 

O’GeneRuler 

DNA Ladders 

MassRuler 

DNA Ladders 

FastRuler 

DNA Ladders 

O’RangeRuler 

DNA Ladders 

ZipRuler Express 

DNA Ladders 

Conventional 

DNA Markers 

Range, bp 

10-20000 

10-48502 


Agarose gel 

resolution 

time 

Composition 

dependent 

10 min- 

1.5 h 

80-10000 10-45 min 

10-10000 8-14 min 

10-6000 

45 min- 

1.5 h 

100-20000 10-20 min 

Features Applications 

Loading Electrophoresis Quantifi- Labeling* with 

Main feature Origin Formulation dyes 

supplied 

Agarose PAGE Fast 

cation 

T4 PNK Klenow 

Formulated 

according 

to customer 

specifications. 

Bulk quantities. 

Sizing and 

approximate 

quantification of 

a wide range of 

double-stranded 

DNA 

Accurate DNA 

quantification 

Fast DNA 

fragment 

separation in 

short run times 

Individual, 

chromatography-purified 

DNA fragments 

Mixtures of 

individual, 


DNA fragments 

Mixtures of 

individual, 


DNA fragments 

Mixtures of 

5 individual, 


DNA fragments 

Ligation 

Step ladders 

products 10, 

composed of 5, 

15, 20, 50, 

10, 20, 50, 100, 

100, 200, 

200, 500 bp DNA 

500 bp DNA 

fragments 

fragments 

Fast and accurate 

sizing of a broad 

range DNA 

fragments 

8-23130 bp 45 min-18 h Traditional 

markers 

Mixtures of 

individual, 


DNA fragments 

Digested 

Lambda, Phage 

and plasmid 

DNA 

Note 

Not all the DNA ladders/markers of the group are suitable for the indicated application. 

* Only DNA ladders/markers supplied in TE buffer can be labeled. 

** Excluding Lambda markers. 

In TE buffer or 

ready-to-use 


ready-to-use 

Ready-to-use 

Ready-to-use 

Ready-to-use 

Ready-to-use 


ready-to-use 

Any Thermo 


Loading Dye 

upon request 

6X DNA 

Loading 

Dye or 

6X Orange 

Loading Dye 

6X MassRuler 

Loading Dye 

6X MassRuler 

Loading Dye, 

6X Orange 

Loading Dye 

6X Orange 

Loading Dye 

6X Orange 

Loading Dye, 

6X MassRuler 

Loading Dye 

6X DNA 

Loading Dye 

Page 

– 370 

– – 358 

– – – 362 

– 364 

– – – – 366 

– – – 365 

** – 368

Range selection for DNA Ladders and Markers 

10-300 bp 

10-200 bp 

10 bp 100 bp 1 kb 10 kb 50 kb 

25-700 bp 

50-1000 bp 

75-20000 bp 

100-1000 bp 

100-3000 bp 

100-5000 bp 

100-10000 bp 

80-1031 bp 

100-1000 bp 

100-1000 bp 

80-10000 bp 

100-10000 bp 

100-10000 bp 

50-1500 bp 

100-5000 bp 

8-587 bp 

26-501 bp 

72-1353 bp 

125-23130 bp 

125-21226 bp 

250-10000 bp 

500-10000 bp 

10-100 bp 

10-150 bp 

20-300 bp 

50-1000 bp 

100-1500 bp 

100-6000 bp 

200-3000 bp 

500-6000 bp 

100-10000 bp 

200-20000 bp 

1500-10000 bp 

1500-10000 bp 

1500-10000 bp 

3530-21226 bp 

10171-48502 bp 

10bp 100bp 1kb 10kb 50kb 

GeneRuler Ultra Low Range DNA Ladder, #SM1211/2/3, p.361 

O’GeneRuler Ultra Low Range DNA Ladder, #SM1223, p.361 

GeneRuler Low Range DNA Ladder, #SM1191/2/3, p.361 

O’GeneRuler Low Range DNA Ladder, #SM1203, p.361 

GeneRuler 50 bp DNA Ladder, #SM0371/2/3, p.360 

O’GeneRuler 50 bp DNA Ladder, #SM1133, p.360 

GeneRuler 1 kb Plus DNA Ladder, #SM1331/2/3/4, p.360 

O’GeneRuler 1 kb Plus DNA Ladder, #SM1343, p.360 

GeneRuler 100 bp DNA Ladder, #SM0241/2/3/4, p.360 

O’GeneRuler 100 bp DNA Ladder, #SM1143, p.360 

GeneRuler 100 bp Plus DNA Ladder, #SM0321/2/3/4, p.360 

O’GeneRuler 100 bp Plus DNA Ladder, #SM1153, p.360 

GeneRuler Express DNA Ladder, #SM1551/2/3, p.361 

O’GeneRuler Express DNA Ladder, #SM1563, p.361 

GeneRuler DNA Ladder Mix, #SM0331/2/3/4, p.360 

O’GeneRuler DNA Ladder Mix, #SM1173, p.360 

GeneRuler 1 kb DNA Ladder, #SM0311/2/3/4, p.360 

O’GeneRuler 1kb DNA Ladder, #SM1163, p.360 

GeneRuler High Range DNA Ladder, #SM1351/2/3, p.361 

MassRuler Low Range DNA Ladder, #SM0383, p.363 

MassRuler Express LR Forward DNA Ladder, #SM1263, p.363 

MassRuler Express LR Reverse DNA Ladder, #SM1273, p.363 

MassRuler DNA Ladder Mix, #SM0403, p.363 

MassRuler Express Forward DNA Ladder Mix, #SM1283, p.363 

MassRuler Express Reverse DNA Ladder Mix, #SM1293, p.363 

MassRuler High Range DNA Ladder, #SM0393, p.363 

MassRuler Express HR Forward DNA Ladder, #SM1243, p.363 

MassRuler Express HR Reverse DNA Ladder, #SM1253, p.363 

FastRuler Ultra Low Range DNA Ladder, #SM1233, p.364 

FastRuler Low Range DNA Ladder, #SM1103, p.364 

FastRuler Middle Range DNA Ladder, #SM1113, p.364 

FastRuler High Range DNA Ladder, #SM1123, p.364 

O’RangeRuler 5 bp DNA Ladder, #SM1303, p.367 





O’RangeRuler 100+500 bp DNA Ladder, #SM0653, p.367 


O’RangeRuler 500bp DNA Ladder, #SM0643, p.367 

ZipRuler Express DNA Ladder 1, #SM1373, p.365 

ZipRuler Express DNA Ladder 2, #SM1373, p.365 

pBR322 DNA/BsuRI Marker, 5, #SM0271, p.369 

pUC19 DNA/MspI Marker, 23, #SM0221/2/3, p.369 

X174 DNA/BsuRI Marker, 9, #SM0251/2/3, p.369 

Lambda DNA/HindIII Marker, 2, #SM0101/2/3, p.369 

Lambda DNA/EcoRI+HindIII Marker, 3, #SM0191/2/3, p.369 

Lambda DNA/EcoRI Marker, 1, #SM0281, p.369 


356 

Reference for DNA Ladders and Markers 

50 kb 

10 kb 

1 kb 

100 bp 

50 kb 

10 kb 

1 kb 

100 bp 

10 bp 

GeneRuler Ultra Low Range 

DNA Ladder #SM1211/2/3, p.361 

O’GeneRuler Ultra Low Range 

DNA Ladder #SM1223, p.361 

MassRuler Low Range 

DNA Ladder 

#SM0383, p.363 


GeneRuler Low Range DNA Ladder 

#SM1191/2/3, p.361 

O’GeneRuler Low Range DNA Ladder 

#SM1203, p.361 

MassRuler Express LR Forward 


MassRuler Express LR Reverse 


GeneRuler 50 bp DNA Ladder 

#SM0371/2/3, p.360 

O’GeneRuler 50 bp DNA Ladder 

#SM133, p.360 

MassRuler DNA 

Ladder Mix 

#SM0403, p.363 

GeneRuler 1 kb Plus DNA Ladder 

#SM1331/2/3, p.360 

O’GeneRuler 1 kb Plus DNA Ladder 

#SM1343, p.360 

MassRuler Express Forward 

DNA Ladder Mix #SM1283, p.363 

MassRuler Express Reverse 

DNA Ladder Mix #SM1293, p.363 


#SM0241/2/3 p.360 

O’GeneRuler 100 bp DNA Ladder 

#SM1143 p.360 

MassRuler High Range 

DNA Ladder 

#SM0393, p.363 

GeneRuler 100 bp Plus DNA Ladder 

#SM0321/2/3, p.360 

O’GeneRuler 100 bp Plus DNA Ladder 

#SM1153 p.360 

MassRuler Express HR Forward 


MassRuler Express HR Reverse 


GeneRuler Express DNA Ladder 

#SM1551/2/3, p.361 

O’GeneRuler Express DNA Ladder 

#SM1563 p.361 

FastRuler Ultra Low Range 

DNA Ladder 

#SM1233, p.364 

GeneRuler DNA Ladder Mix 

#SM0331/2/3, p.360 

O’GeneRuler DNA Ladder Mix 

#SM1173, p.360 

FastRuler Low Range 

DNA Ladder 

#SM1103, p.364 

GeneRuler 1 kb DNA Ladder 

#SM0311/2/3, 

p.360O’GeneRuler 1 kb DNA 

Ladder 

#SM1163, p.360 

10 bp 10 bp 

FastRuler Middle Range 

DNA Ladder 

#SM1113, p.364 

GeneRuler High Range 

DNA Ladder 

#SM1351/2/3, p.361 

FastRuler High Range 

DNA Ladder 

#SM1123, p.364 

50 kb 

10 kb 

1 kb 

100 bp 

50 kb 

10 kb 

1 kb 

100 bp 

10 bp

50 kb 

10 kb 

1 kb 

100 bp 

10 bp 

50 kb 

10 kb 

1 kb 

100 bp 

10 bp 

O’RangeRuler 5 bp 

DNA Ladder 

#SM1303, p.367 

pBR322 DNA/BsuRI, 5 

#SM0271, p.369 


DNA Ladder 

#SM1313, p.367 

pUC19 DNA/MspI, 23 

#SM0221/2/3, p.369 


DNA Ladder 

#SM1323, p.367 

X174 DNA/BsuRI, 9 

#SM0251/2/3, p.369 


DNA Ladder 

#SM0613, p.367 

50 kb 

10 kb 

1 kb 

100 bp 

10 bp 


DNA Ladder 

#SM0623, p.367 

* Indicates fragments with cohesive ends of the 12 nt cos sites of DNA that may anneal and form an additional band. These fragments (shown in italic) can be separated by h eating at 65°C 

for 5 min and then cooling on ice for 3 min. 

Indicates a possible additional band due to annealing of cohesive ends from 12 nt cos sites of DNA. Reference bands appear in red. 

O’RangeRuler 100+500 bp 

DNA Ladder 

#SM0653, p.367 


DNA Ladder 

#SM0633, p.367 

50 kb 

10 kb 

1 kb 

100 bp 

10 bp 


DNA Ladder 

#SM0643, p.367 

Lambda DNA/HindIII, 2 

#SM0101/2/3, p.369 

ZipRuler Express DNA 

Ladder 1 

#SM1373, p.365 

Lambda DNA/EcoRI+HindIII, 3 

#SM0191/2/3, p.369 

ZipRuler Express DNA 

Ladder 2 

#SM1373, p.365 

Lambda DNA/EcoRI, 1 

#SM0281, p.369 

50 kb 

10 kb 

1 kb 

100 bp 

10 bp 

50 kb 

10 kb 

1 kb 

100 bp 

10 bp 


358 

Products 

GeneRuler and O’GeneRuler DNA Ladders (10-20000 bp) 


TopVision Agaroses p.427 

Loading Dyes p.372 




ATP p.411 

0.5 M EDTA, pH 8.0 p.421 



» 7.1. General recommendations for DNA 

electrophoresis p.374 

» 7.2. Preparation of agarose gels for DNA 


» 7.3. Preparation of gels for PAGE p.375 

» 7.4. Preparation of DNA samples for 


» 7.5. Preparation of DNA ladders/markers 

for electrophoresis p.376 

» 7.7. Separation of Express DNA ladders in 

different electrophoresis conditions p.377 

» 6.1. DNA/RNA end labeling p.349 


Description 

Thermo Scientific GeneRuler DNA ladders are 

mixtures of chromatography-purified individual 


The GeneRuler line includes the most popular 

ladders such as the 100 bp and 1 kb DNA 

Ladder. 

GeneRuler DNA ladders are available in 

two formats: conventional (TE buffer) and a 

ready-to-use format (premixed with 6X DNA 

Loading Dye which contains bromophenol blue 

and xylene cyanol FF). 

Conventional versions can be labeled 

radioactively with T4 Polynucleotide 

Kinase (#EK0031). 

O’GeneRuler DNA ladders are another 

ready-to-use version of GeneRuler DNA ladders. 

O’GeneRuler DNA ladders are premixed with 

6X Orange DNA Loading Dye, which contains 

xylene cyanol FF and orange G. In a 1% agarose 

gel orange G dye migrates at 50 bp. Therefore 

O’GeneRuler DNA ladders are ideal to use 

when visualization of small DNA fragments is 

important. 

GeneRuler Ultra Low Range and Low 

Range DNA ladders are ideal for approximate 

quantification and sizing of small molecular 

weight DNA fragments. The ladders can 

be used in agarose and nondenaturing 

polyacrylamide gel electrophoresis 

(see Figure p.375). 

GeneRuler and O’GeneRuler Ultra Low 

Range DNA ladders can also be used for 

analysis of siRNA. 

The GeneRuler High Range DNA Ladder 

is designed for fast sizing of h igh 

molecular weight DNA fragments 

(1.5 h in 0.4% agarose gel). 

GeneRuler and O’GeneRuler Express DNA 

ladders are designed for fast separation (in 

5-15 min at 23 V/cm) under a wide range of 

electrophoresis conditions, in different buffers, 

voltages or gel percentages. 

The ladders are supplied (depending on the 

version) either with 6X DNA Loading Dye or with 

6X Orange DNA Loading Dye for sample DNA. 

Features 

Ideal for both DNA sizing and approximate 

quantification. 

Sharp bands. 

Composed of chromatography purified 

individual DNA fragments. 

Bright reference bands. 

Ready-to-use ladders can be direct loaded 

on gels and are stable at room temperature 

for 6 months. 

Supplied with loading dye for sample DNA. 

Storage Buffer (TE buffer) 


GeneRuler Storage and Loading Buffer 

(for ready-to-use ladders) 

10 mM Tris-HCl (pH 7.6), 10 mM EDTA, 

0.005% bromophenol blue, 0.005% xylene 

cyanol FF and 10% glycerol. 

6X DNA Loading Dye 

10 mM Tris-HCl (pH 7.6), 0.03% bromophenol 

blue, 0.03% xylene cyanol FF, 60% glycerol and 

60 mM EDTA. 

O’GeneRuler Storage and Loading Buffer 


0.025% orange G, 0.005% xylene cyanol FF 

and 10% glycerol. 

6X Orange DNA Loading Dye 

10 mM Tris-HCl (pH 7.6), 0.15% orange G, 

0.03% xylene cyanol FF, 60% glycerol and 

60 mM EDTA. 

Storage 


Ready-to-use versions can be stored at room 

temperature or at 4°C for 6 months.

DNA ladder Cat. # 

Concentration, 

μg/μl 

Amount, 

μg 

Applications, 

0.5 μg/lane 

Loading, 

μg(μl)/lane 


SM0311 

SM0312 

0.5 

250 (5x50) 

1250 (25x50) 

500 

2500 

0.5 (1) 

GeneRuler 1 kb DNA Ladder, SM0313 

250 (5x50) 500 

ready-to-use 

O’GeneRuler 1 kb DNA Ladder, 

ready-to-use 

SM0314 

SM1163 

0.1 

50 

250 (5x50) 

100 

500 

0.5 (5) 


SM1331 

SM1332 

0.5 

250 (5x50) 

1250 (25x50) 

500 

2500 

0.5 (1) 

GeneRuler 1 kb Plus DNA Ladder, SM1333 

250 (5x50) 500 

ready-to-use 

O’GeneRuler 1 kb Plus DNA Ladder, 

ready-to-use 

SM1334 

SM1343 

0.1 

50 

250 (5x50) 

100 

500 

0.5 (5) 


SM0331 

SM0332 

0.5 

250 (5x50) 

1250 (25x50) 

500 

2500 

0.5 (1) 

GeneRuler DNA Ladder Mix, SM0333 

250 (5x50) 500 

ready-to-use 

O’GeneRuler DNA Ladder Mix, 

ready-to-use 

SM0334 

SM1173 

0.1 

50 

250 (5x50) 

100 

500 

0.5 (5) 


SM0241 

SM0242 

0.5 

50 

250 (5x50) 

100 

500 

0.5 (1) 

GeneRuler 100 bp DNA Ladder, SM0243 

50 100 

ready-to-use 

O’GeneRuler 100 bp DNA Ladder, 

ready-to-use 

SM0244 

SM1143 

0.1 

250 (5x50 

50 

500 

100 

0.5 (5) 

GeneRuler 100 bp Plus DNA Ladder SM0321 

SM0322 

0.5 

50 

250 (5x50) 

100 

500 

0.5 (1) 

GeneRuler 100 bp Plus DNA Ladder, SM0323 

50 100 

ready-to-use 

O’GeneRuler 100 bp Plus DNA 

Ladder, ready-to-use 

SM0324 

SM1153 

0.1 

250 (5x50) 

50 

500 

100 

0.5 (5) 


SM0371 

SM0372 

0.5 

50 

250 (5x50) 

100 

500 

0.5 (1) 

GeneRuler 50 bp DNA Ladder, 

ready-to-use 


ready-to-use 

SM0373 

SM1133 

0.1 50 100 0.5 (5) 

GeneRuler Ultra Low Range DNA 

Ladder 

SM1211 

SM1212 

0.5 

50 

250 (5x50) 

50-100 

250-500 

0.5-1 (1-2) 

GeneRuler Ultra Low Range DNA 


O’GeneRuler Ultra Low Range DNA 


SM1213 

SM1223 

0.1 50 100 0.5-1 (5-10) 

GeneRuler Low Range DNA Ladder SM1191 

SM1192 

0.5 

50 

250 (5x50) 

50-100 

250-500 

0.5-1 (1-2) 

GeneRuler Low Range DNA Ladder, 

ready-to-use 

O’GeneRuler Low Range DNA 


SM1193 

SM1203 

0.1 50 100 0.5-1 (5-10) 

GeneRuler High Range DNA Ladder SM1351 

SM1352 

0.5 

50 

250 (5x50) 

100 

500 

0.5 (1) 

GeneRuler High Range DNA Ladder, 

ready-to-use 

SM1353 0.1 50 100 0.5 (5) 


SM1551 

SM1552 

0.5 

50 

250 (5x50) 

100 

500 

0.5 (1) 

GeneRuler Express DNA Ladder, 

ready-to-use 

O’GeneRuler Express DNA Ladder, 

ready-to-use 

SM1553 

SM1563 

0.1 50 100 0.5 (5) 


6X DNA Loading Dye 1 ml/2 ml/10 ml 

6X Orange DNA Loading Dye 1 ml/2 ml/10 ml 

Range, 

bp 

Number of 

fragments 

Agarose, 

% 

PAGE, 

% 

250-10000 14 0.7-1.2 – 

75-20000 15 0.7-1.2 – 

100-10000 21 0.7-1.2 – 

100-1000 10 1.7-2.5 4-8 

100-3000 14 1.7-2.5 – 

50-1000 13 1.7-2.5 4-8 

10-300 11 4.5-5.0 8-10 

25-700 10 2.5-3.0 8-10 

10171- 

48502 

8 0.3-0.5 – 

100-5000 9 1.7-2.5 – 


360 


#SM0311/2/3 

O’GeneRuler 1 kb DNA Ladder, 

ready-to-use 

#SM1163 

1% TopVision Agarose (#R0491) 


#SM0241/2/3 


ready-to-use 

#SM1143 

1.7% TopVision Agarose (#R0491) 

bp ng/0.5 μg % 

10000 30.0 6.0 

8000 30.0 6.0 

6000 70.0 14.0 

5000 30.0 6.0 

4000 30.0 6.0 

3500 30.0 6.0 

3000 70.0 14.0 

2500 25.0 5.0 

2000 25.0 5.0 

1500 25.0 5.0 

1000 60.0 12.0 

750 25.0 5.0 

500 25.0 5.0 

250 25.0 5.0 

0.5 μg/lane, 8 cm length gel, 

1X TAE, 7 V/cm, 45 min 


1000 

900 

800 

45.0 

45.0 

45.0 

9.0 

9.0 

9.0 

700 45.0 9.0 

600 

500 

45.0 

115.0 

9.0 

23.0 

400 40.0 8.0 

300 40.0 8.0 

200 40.0 8.0 

100 40.0 8.0 


1X TBE, 5 V/cm, 1 h 


5% polyacrylamide 

bp 

1000 

900 

800 

700 

600 

500 

400 

300 

200 

100 


1X TAE, 8 V/cm, 3 h 


#SM1331/2/3 

O’GeneRuler 1 kb Plus DNA Ladder, 

ready-to-use 

#SM1343 


GeneRuler 100 bp Plus DNA Ladder 

#SM0321/2/3 

O’GeneRuler 100 bp Plus DNA Ladder, 

ready-to-use 

#SM1153 



20000 20.0 4.0 

10000 20.0 4.0 

7000 

5000 

4000 

3000 

20.0 

75.0 

20.0 

20.0 

4.0 

15.0 

4.0 

4.0 

2000 20.0 4.0 

1500 80.0 16.0 

1000 25.0 5.0 

700 25.0 5.0 

500 

400 

75.0 

25.0 

15.0 

5.0 

300 

200 

25.0 

25.0 

5.0 

5.0 

75 25.0 5.0 




3000 28.0 5.6 

2000 28.0 5.6 

1500 

1200 

28.0 

28.0 

5.6 

5.6 

1000 

900 

80.0 

27.0 

16.0 

5.4 

800 27.0 5.4 

700 27.0 5.4 

600 

500 

27.0 

80.0 

5.4 

16.0 

400 30.0 6.0 

300 30.0 6.0 

200 30.0 6.0 

100 30.0 6.0 




bp 

3000 

2000 

1500 

1200 

1000 

900 

800 

700 

600 

500 

400 

300 

200 

100 




#SM0331/2/3 

O’GeneRuler DNA Ladder Mix, 

ready-to-use 

#SM1173 



10000 18.0 3.6 

8000 18.0 3.6 

6000 18.0 3.6 

5000 18.0 3.6 

4000 18.0 3.6 

3500 18.0 3.6 

3000 60.0 12.0 

2500 16.0 3.2 

2000 16.0 3.2 

1500 16.0 3.2 

1200 16.0 3.2 

1000 

900 

800 

700 

600 

500 

400 

60.0 

17.0 

17.0 

17.0 

17.0 

60.0 

20.0 

12.0 

3.4 

3.4 

3.4 

3.4 

12.0 

4.0 

300 20.0 4.0 

200 

100 

20.0 

20.0 

4.0 

4.0 



GeneRuler 50bp DNA Ladder 

#SM0371/2/3 

O’GeneRuler 50bp DNA Ladder, 

ready-to-use 

#SM1133 


bp ng/0.5μg % 

1000 

900 

30.0 

30.0 

6.0 

6.0 

800 

700 

600 

500 

30.0 

30.0 

30.0 

75.0 

6.0 

6.0 

6.0 

15.0 

400 30.0 6.0 

300 30.0 6.0 

250 75.0 15.0 

200 35.0 7.0 

150 35.0 7.0 

100 35.0 7.0 

50 35.0 7.0 




bp 

1000 

900 

800 

700 

600 

500 

400 

300 

250 

200 

150 

100 

50 


1X TAE, 8 V/cm, 3 h

GeneRuler Ultra Low Range DNA Ladder 

#SM1211/2/3 

O’GeneRuler Ultra Low Range DNA Ladder, 

ready-to-use 

#SM1223 



#SM1551/2/3 

O’GeneRuler Express DNA Ladder, 

ready-to-use 

#SM1563 



300 30.0 6.0 

200 32.5 6.5 

150 35.0 7.0 

100 37.5 7.5 

75 37.5 7.5 

50 105.0 21.0 

35 37.5 7.5 

25 37.5 7.5 

20 40.0 8.0 

15 47.5 9.5 

10 60.0 12.0 




5000 40.0 8.0 

3000 40.0 8.0 

2000 40.0 8.0 

1500 100.0 20.0 

1000 40.0 8.0 

750 40.0 8.0 

500 100.0 20.0 

300 50.0 10.0 

100 50.0 10.0 




bp 

300 

200 

150 

100 

75 

50 

35 

25 

20 

15 

10 

0.5 μg/lane, 20c m length gel, 


GeneRuler Low Range DNA Ladder 

#SM1191/2/3 

O’GeneRuler Low Range DNA Ladder, 

ready-to-use 

#SM1203 


5 min 


700 30.0 6.0 

500 32.5 6.5 

400 35.0 7.0 

300 90.0 18.0 

200 35.0 7.0 

150 35.0 7.0 

100 90.0 18.0 

75 37.5 7.5 

50 40.0 8.0 

25 75.0 15.0 



10 min 

15 min 


bp 

700 

500 

400 

300 

200 

150 

100 

75 

Fast separation of Thermo Scientific GeneRuler 

Express DNA Ladder at h igh voltage (23 V/cm). 

Electrophoresis conditions: 0.5 μg/lane, 1% TopVision 

Agarose (#R0491), 1X TAE, 23 V/cm, 5-15 min. 

50 

25 

0.5 μg/lane, 2 0 cm length gel, 


GeneRuler High Range DNA Ladder 

#SM1351/2/3 



48502 80.0 16.0 

24508 88.0 17.7 

20555 80.0 15.9 

17000 59.0 11.8 

15258 59.0 11.8 

13825 50.0 10.0 

12119 47.0 9.4 

10171 37.0 7.3 


1X TAE, 3 V/cm, 1.5 h 


362 

MassRuler DNA Ladders, ready-to-use (80-10000 bp) 

DNA ladder, ready-to-use Cat. # 


ng/μl 

MassRuler Low Range DNA Ladder SM0383 60.8 

MassRuler Express LR Forward DNA Ladder SM1263 

MassRuler Express LR Reverse DNA Ladder SM1273 

MassRuler High Range DNA Ladder SM0393 42.2 

MassRuler Express HR Forward DNA Ladder SM1243 

MassRuler Express HR Reverse DNA Ladder SM1253 

MassRuler DNA Ladder Mix SM0403 103 

MassRuler Express Forward DNA Ladder Mix SM1283 

MassRuler Express Reverse DNA Ladder Mix SM1293 


6X MassRuler DNA Loading Dye 1 ml Description 









electrophortesis p.374 

» 7.1.1. Recommendations for accurate in-gel 

quantification p.374 





» 7.7. Separation of Express DNA ladders 

in different electrophoresis conditions p.377 


Volume, 

μl 

Applications 

Loading, 

μl/lane 

Range, 

bp 

Number of 

fragments 

Agarose, 

% 

80-1031 11 1.7-2.5 

28 

2x500 50-200 5-20 

100-1000 6 0.7-2.0 

9 0.7-1.2 

28.5 

2x500 50-200 5-20 1500-10000 

6 0.7-1.5 

80-10000 20 1.0-1.2 

56.5 

2x500 50-200 5-20 

100-10000 12 0.7-1.5 

Thermo Scientific MassRuler DNA ladders are 

specially designed for accurate quantification 

and sizing of DNA fragments by agarose gel 

electrophoresis. The intensity of the fragment in 

each ladder is calibrated against a standard that 

guarantees the precise quantity of each band. 

The ladders are mixtures of chromatographypurified, 


MassRuler Express Forward and Reverse 

DNA Ladders allow fast and reliable DNA 

quantification in short separation distances 

under various electrophoresis conditions. In 

the forward versions, the mass of each DNA 

fragment is directly proportional to the fragment 

size, whereas in the reverse versions, the mass 

is inversely proportional to the fragment’s size. 

Features 

Accurate DNA sizing and quantification. 

Sharp bands. 

Assembled of chromatography purified 


Easy-to-remember fragment sizes and 

quantities. 

Ready-to-use – premixed with Loading Dye. 


Storage and Loading Buffer 


0.005% bromophenol blue, 10% glycerol. 

6X MassRuler DNA Loading Dye 


blue, 60% glycerol and 60 mM EDTA. 

Storage 

Store at -20°C (or at 4°C or room temperature 

for 6 months). 

A. MassRuler Express LR Forward DNA Ladder B. MassRuler Express LR Reverse DNA Ladder 

[FU] 

[FU] 

5000 

5000 

4000 

4000 

3000 

3000 

2000 

2000 

1000 

1000 

0 

0 

40 60 80 100 

40 60 80 100 

Analysis of the Thermo Scientific MassRuler Express LR Forward and Reverse DNA Ladders 

(#SM1263 and #SM1273) using the Agilent 2100 Bioanalyzer DNA 1000 LabChip Kit. 

A – analysis of MassRuler Express LR Forward DNA Ladder 

B – analysis of MassRuler Express LR Reverse DNA Ladder

MassRuler Low Range DNA Ladder, 

ready-to-use 

#SM0383 


10 μl/lane, 8 cm length gel, 

1X TBE, 5 V/cm, 1.5 h 

bp ng/20 μl ng/15 μl ng/10 μl ng/5 μl 

1031 

900 

800 

700 

600 

200 

180 

160 

140 

120 

150 

135 

120 

105 

90 

100 

90 

80 

70 

60 

50 

45 

40 

35 

30 

500 200 150 100 50 

400 80 60 40 20 

300 60 45 30 15 

200 40 30 20 10 

100 20 15 10 5 

80 16 2 8 4 

MassRuler High Range DNA Ladder, 

ready-to-use 

#SM0393 





10000 200 150 100 50 

8000 160 120 80 40 

6000 120 90 60 30 

5000 100 75 50 25 

4000 80 60 40 20 

3000 60 45 30 15 

2500 52 39 26 13 

2000 40 30 20 10 

1500 32 24 16 8 

MassRuler DNA Ladder Mix, 

ready-to-use 

#SM0403 



10000 200 

8000 160 

6000 120 

5000 100 

4000 80 

3000 60 

2500 52 

2000 40 

1500 32 

150 

120 

90 

75 

60 

45 

39 

30 

24 

100 

80 

60 

50 

40 

30 

26 

20 

16 

50 

40 

30 

25 

20 

15 

13 

10 

8 

1031 

900 

800 

700 

600 

500 

400 

200 

180 

160 

140 

120 

200 

80 

150 

135 

120 

105 

90 

150 

60 

100 

90 

80 

70 

60 

100 

40 

50 

45 

40 

35 

30 

50 

20 

300 60 45 30 15 

200 40 30 20 10 

100 

80 

20 

16 

15 

12 

10 

8 

5 

4 



MassRuler Express LR Forward DNA Ladder, ready-to-use 

#SM1263 

MassRuler Express LR Reverse DNA Ladder, ready-to-use 

#SM1273 

ng/20 μl ng/15 μl ng/10 μl ng/5 μl bp 

200 150 100 50 1000 

140 105 70 35 700 

100 75 50 25 500 

60 45 30 15 300 

40 30 20 10 200 

20 15 10 5 100 

MassRuler Express HR Forward DNA Ladder, ready-to-use 

#SM1243 

MassRuler Express HR Reverse DNA Ladder, ready-to-use 

#SM1253 


200 150 100 50 10000 

140 

100 

105 

75 

70 

50 

35 

25 

7000 

5000 

60 45 30 15 3000 

40 30 20 10 2000 

30 22.5 15 7.5 1500 

MassRuler Express Forward DNA Ladder Mix, ready-to-use 

#SM1283 

MassRuler Express Reverse DNA Ladder Mix, ready-to-use 

#SM1293 


200 150 100 50 10000 

140 

100 

105 

75 

70 

50 

35 

25 

7000 

5000 

60 45 30 15 3000 

40 30 20 10 2000 

30 22.5 15 7.5 1500 

200 150 100 50 1000 

140 105 70 35 700 

100 75 50 25 500 

60 45 30 15 300 

40 30 20 10 200 

20 15 10 5 100 

Forward Reverse 




1000 20 15 10 5 

700 40 30 20 10 

500 60 45 30 15 

300 100 75 50 25 

200 140 105 70 35 

100 200 150 100 50 


10 μl/lane, 8 cm length gel, 1X TAE, 7 V/cm, 30 min 


10000 

7000 

5000 

30 

40 

60 

22.5 

30 

45 

15 

20 

30 

7.5 

10 

15 

3000 100 75 50 25 

2000 140 105 70 35 

1500 200 150 100 50 




10000 

7000 

5000 

30 

40 

60 

22.5 

30 

45 

15 

20 

30 

7.5 

10 

15 

3000 100 75 50 25 

2000 140 105 70 35 

1500 200 150 100 50 

1000 20 15 10 5 

700 40 30 20 10 

500 60 45 30 15 

300 100 75 50 25 

200 140 105 70 35 

100 200 150 100 50 




364 

FastRuler DNA Ladders, ready-to-use (10-10000 bp) 



ng/μl 

FastRuler Ultra Low Range DNA Ladder SM1233 22.2 


Volume, 

μl 

Description 

Thermo Scientific FastRuler DNA ladders 

are specifically designed for fast sizing and 

quantification of double-stranded DNA in 

48-well (or 96-well) h igh throughput gels, 

as well as in conventional agarose gels. 

These ladders are mixtures of five blunt-end 

chromatography-purified individual DNA 

fragments that are easily resolved in a short 

separation distance (10-20 mm) after an 

8-14 min run. The ladders are especially 

useful for electrophoresis of PCR products. 

The FastRuler Ultra Low Range DNA Ladder 

contains dephosphorylated DNA fragments 

and is ideal for 5’-end labeling with T4 

Polynucleotide Kinase (#EK0031) 

in the forward reaction. 

Features 

Fast separation (8-14 min). 

Short separation distance (10-20 mm). 

Ideal for PCR products. 

Sharp bands. 

Easy-to-remember fragment sizes and 

quantities. 

Ready-to-use – premixed with loading dye. 

Supplied with loading dye solution 

for sample DNA. 

FastRuler Ultra Low Range DNA Ladder, 

ready-to-use 

#SM1233 

bp ng/20 μl ng/15 μl ng/10 μl ng/5 μl ng/3 μl 

200 80 60 40 20 12 

100 

50 

80 

80 

60 

60 

40 

40 

20 

20 

12 

12 

20 80 60 40 20 12 

10 124 93 62 31 19 


20 μl/lane, 1X TBE, 7 V/cm, 14 min 

Applications 

FastRuler Middle Range DNA Ladder, 

ready-to-use 

#SM1113 

bp ng/20 μl ng/15μl ng/10 μl ng/5 μl ng/3 μl 

5000 80 60 40 20 12 

2000 80 60 40 20 12 

850 80 60 40 20 12 

400 80 60 40 20 12 

100 80 60 40 20 12 


20 μl/lane, 1X TAE, 7 V/cm, 14 min 

Loading, 

μl/lane 

Range, 

bp 

Number of 

fragments 

10-200 

4.0 

FastRuler Low Range DNA Ladder 

FastRuler Middle Range DNA Ladder 

SM1103 

SM1113 

20 

20 

2x500 50-333 3-20 

50-1500 

100-5000 

5 

2.0 

1.0 

FastRuler High Range DNA Ladder SM1123 20 500-10000 1.0 


6X MassRuler DNA Loading Dye 1 ml 

or 

6X Orange DNA Loading Dye 1 ml 







ATP p.411 

0.5 M EDTA, pH 8.0 p.421 



» 6.1.2. DNA/RNA 5’-end labeling by 

T4 PNK in the forward reaction p. 349 













(for FastRuler Ultra Low Range) 


0.025% orange G, 0.005% xylene cyanol FF, 

10% glycerol. 

6X MassRuler DNA Loading Dye 


blue, 60% glycerol and 60 mM EDTA. 




60 mM EDTA. 

Storage 



FastRuler Low Range DNA Ladder, 

ready-to-use 

#SM1103 


1500 

850 

80 

80 

60 

60 

40 

40 

20 

20 

12 

12 

400 80 60 40 20 12 

200 80 60 40 20 12 

50 80 60 40 20 12 


20 μl/lane, 1X TBE, 7 V/cm, 14 min 

FastRuler High Range DNA Ladder, 

ready-to-use 

#SM1123 


10000 80 60 40 20 12 

4000 80 60 40 20 12 

2000 80 60 40 20 12 

1000 80 60 40 20 12 

500 80 60 40 20 12 


20 μl/lane, 1X TAE, 7 V/cm, 14 min 

Agarose, 

%

ZipRuler Express DNA Ladder Set, ready-to-use (100-20000 bp) 


ZipRuler Express DNA Ladder Set 

ZipRuler Express DNA Ladder 1 

ZipRuler Express DNA Ladder 2 


6X Orange DNA Loading Dye 1 ml 







% ng/0.5 μg bp 

8.8 44 10000 

25.6 128 5000 

8.8 44 3000 

8.8 44 2000 

8.0 40 1200 

8.0 40 850 

16.0 80 500 

8.0 40 300 

8.0 40 100 

1 2 

20 min 


μg/μl 

Amount, 

μg 

Applications 

Description 

The Thermo Scientific ZipRuler Express 

DNA Ladder Set contains two ladders: 

ZipRuler Express DNA Ladder 1 and ZipRuler 

Express DNA Ladder 2. Both are mixtures 

of chromatography-purified individual DNA 

fragments. The set is designed for fast and 

accurate sizing of a broad range of DNA 

fragments and is compatible with various 

electrophoresis conditions. 

For fast separation, the two ladders are loaded 

into two different wells of the same gel. For 

longer runs, equal amounts of both ladders are 

loaded into the same gel well. 

ZipRuler Express DNA Ladder 1 is premixed 

with the 6X Orange DNA Loading Dye 

(orange G and xylene cyanol), whereas 

ZipRuler Express DNA Ladder 2 is premixed 

with the 6X MassRuler DNA Loading Dye 

(bromophenol blue). When both ladders are 

loaded in the same gel lane, the migration of 

DNA is monitored by three electrophoresis 

tracking dyes; bromophenol blue, xylene cyanol 

FF and orange G. 

Features 

Flexible – can be run in two lanes or 

combined in one lane. 

Composed of chromatography purified 


Ready-to-use – premixed with loading dye for 

direct loading on gels. 


Loading, 

μg(μl)/lane 

Range, 

bp 

SM1373 0.1 2X50 100-200 0.25-0.5 (2.5-5) 100-10000 

200-20000 

ZipRuler Express DNA Ladder Set, ready-to-use, 

#SM1373 


20000 48 9.6 

7000 48 9.6 

4000 46 9.2 

2500 46 9.2 

1500 152 30.4 

1000 40 8.0 

700 40 8.0 

400 40 8.0 

200 40 8.0 



Mix 

20 min 

% ng/0.5 μg bp 

8.8 44 10000 

25.6 128 5000 

8.8 44 3000 

8.8 44 2000 

8.0 40 1200 

8.0 40 850 

16.0 80 500 

8.0 40 300 

8.0 40 100 

1 2 

40 min 


20000 48 9.6 

7000 48 9.6 

4000 46 9.2 

2500 46 9.2 

1500 152 30.4 

1000 40 8.0 

700 40 8.0 

400 40 8.0 

200 40 8.0 



Number of 

fragments 

Agarose, 

% 

9 5.0 


(for ZipRuler Express DNA Ladder 1) 



10% glycerol. 


(for ZipRuler Express DNA Ladder 2) 






60 mM EDTA. 

Storage 

Store -20°C (or at 4°C or at room temperature 

for up to 6 months). 




» 7.2. Preparation of agarose gels for 

DNA electrophoresis p.374 



» 7.7. Separation of Express DNA ladders 

in different electrophoresis conditions p.377 

Mix 

40 min 

bp 

20000 

10000 

7000 

5000 

4000 

3000 

2500 

2000 

1500 

1200 

1000 

850 

700 

500 

400 

300 

200 

100 

1 – ZipRuler Express 

DNA Ladder 1 

2 – ZipRuler Express 

DNA Ladder 2 

Mix – Ladder 1 and 

Ladder 2 mixed in 

equal volumes 


366 

O’RangeRuler DNA Ladders, ready-to-use (10-6000 bp) 



6X Orange DNA Loading Dye 1 ml Description 









μg/μl 

Amount, 

μg 

Applications 

Loading, 

μg(μl)/lane 

Thermo Scientific O’RangeRuler DNA ladders 

are designed for precise sizing of PCR products 

and other double-stranded DNA fragments in 

agarose or non-denaturing polyacrylamide gels. 

These step ladders consist of ligated blunt end 

basic unit repeats of 5, 10, 20, 50, 100, 200 

or 500 bp. They are not recommended for DNA 

quantification. 

O’RangeRuler DNA ladders are supplied with 

6X Orange DNA Loading Dye for sample DNA. 

The Orange G dye migrates at 50 bp in a 

1% agarose gel and can be used to monitor 

DNA migration in agarose or polyacrylamide 

gels. Orange G is the recommended dye 

to use when visualization of small DNA 

fragments is important. 

Recommended 

Range, bp 

Number of 

fragments 

Agarose, 

% 

O’RangeRuler 5 bp DNA Ladder SM1303 

10-100 19 5.0 

O’RangeRuler 10 bp DNA Ladder SM1313 0.1 50 50-100 0.5-1 (5-10) 10-150 15 4.5-5.0 8-10 

O’RangeRuler 20 bp DNA Ladder SM1323 20-300 15 3.5-4.0 

O’RangeRuler 50 bp DNA Ladder SM0613 

50-1000 20 1.7-2.5 4-8 

O’RangeRuler 100 bp DNA Ladder SM0623 100-1500 15 1.7-2.5 4-8 

O’RangeRuler 200 bp DNA Ladder 

O’RangeRuler 500 bp DNA Ladder 

O’RangeRuler 100+500 bp DNA 

Ladder 

SM0633 

SM0643 

SM0653 

0.05 25 100 0.25 (5) 

200-3000 

500-6000 

100-6000 

15 

12 

32 

0.8-1.2 

0.8-1.2 

1.0-1.2 

– 











PAGE, 

% 

Features 

Step ladders of 5, 10, 20, 50, 100, 200 or 

500 bp increments. 

Sharp bands. 

Easy-to-remember fragment sizes. 

Evenly spaced reference bands. 

Ready-to-use – premixed with 6X Orange 

Loading Dye. 





10% glycerol. 




60 mM EDTA. 

Storage 


for 6 months).


DNA Ladder, ready-to-use 

#SM1303 


bp 

100 

35 

30 

25 

20 

15 

40 

50 

45 

10 

1 μg/lane, 

10 cm length gel, 



bp 

1500 

1000 

900 

800 

700 

600 

500 

400 

300 

200 

100 

0.25 μg/lane, 


1X TBE, 5 V/cm, 1.5 h 


bp 

100 

50 

45 

40 

35 

30 

25 

20 

15 

10 

0.5 μg/lane, 





#SM0623 


bp 

1500 

1000 

900 

800 

700 

600 

500 

400 

300 

200 

100 






#SM1313 


bp 

150 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

1 μg/lane, 




bp 


bp 

150 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0.5 μg/lane, 





#SM0633 

3000 

2800 

2600 

2400 

2200 

2000 

1800 

1600 

1400 

1200 

1000 

800 

600 

400 

200 






#SM1323 




#SM0643 


bp 

300 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

1 μg/lane, 




bp 

6000 

5500 

5000 

4500 

4000 

3500 

3000 

2500 

2000 

1500 

1000 

500 




bp 

300 

200 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0.5 μg/lane, 





#SM0613 


O’RangeRuler 100+500 bp 


#SM0653 


bp 

1000 

500 

450 

400 

350 

300 

250 

200 

150 

100 

50 



1X TBE, 5 V/cm, 1.5 h 


bp 

6000 

5500 

5000 

4500 

4000 

3500 

3000 

2500 

2000 

1500 

1400 

1300 

1200 

1100 

1000 

900 

800 

700 

600 

500 

400 

300 

200 

100 



1X TAE, 7 V/cm,1 h 

bp 

1000 

500 

450 

400 

350 

300 

250 

200 

150 

100 

www.thermoscientific.com/onebio 367 

50 



1X TAE, 8 V/cm, 3 h

368 

Conventional DNA Markers (8-23130 bp) 

DNA marker, marker # Cat. # 


6X DNA Loading Dye 2 ml / 10 ml Description 

The Thermo Scientific Conventional DNA 







Klenow Fragment, exo – p.248 


Modified dNTPs p.413 


ATP p.411 

0.5 M EDTA, pH 8.0 p.421 



» 6.1. DNA/RNA end labeling p.349 














μg/μl 

Amount, 

μg 

Applications, 

0.5 g/lane 

markers are traditional molecular weight 

standards used for sizing and approximate 

quantification of linear double-stranded DNA 

fragments in agarose and non-denaturing 

polyacrylamide gels. The markers are composed 

of lambda, phage or plasmid DNA digested 

to completion with the appropriate restriction 

enzyme(s), purified and dissolved in storage 

buffer. The DNA fragments contain blunt or 

sticky ends depending 

on the restriction enzyme used for the 

marker’s preparation. 

Lambda DNA/HindIII, Lambda DNA/EcoRI+HindIII, 

PhiX174 DNA/BsuRI and pUC19 DNA/MspI 

markers are available in ready-to-use versions – 

premixed with 6X DNA Loading Dye for direct 

loading on agarose gels. 

Markers supplied in TE storage buffer can be 

labeled radioactively with T4 Polynucleotide 

Kinase (#EK0031). Alternatively Lambda and 

pUC19 DNA/MspI markers can be labeled 

radioactively or non-radioactively with Klenow 

Fragment (#EP0051) or Klenow Fragment, 

exo – (#EP0421) using the fill-in reaction. 

Features 

Sizing and approximate quantification of 


Sharp bands. 

Ready-to-use versions are premixed with 6X 

DNA Loading Dye. 


Loading, 

μg(μl)/lane 

Range, 

bp 

Number of 

fragments 

Agarose, 

% 

Lambda DNA/EcoRI Marker, 1 SM0281 0.5 250 (5x50) 500 0.5 (1) 3530-21226 6 0.7 - 

Lambda DNA/HindIII Marker, 2 

Lambda DNA/HindIII Marker, 2, 

ready-to-use 

SM0101 

SM0102 

SM0103 

0.5 

0.1 

250 (5x50) 

1250 (25x50) 

250 (5x50) 

500 

2500 

500 

0.5 (1) 

0.5 (5) 

125-23130 8 1.0 - 

Lambda DNA/EcoRI+HindIII Marker, 3 

Lambda DNA/EcoRI+HindIII Marker, 3, 

ready-to-use 

SM0191 

SM0192 

SM0193 

0.5 

0.1 

250 (5x50) 

1250 (25x50) 

250 (5x50) 

500 

2500 

500 

0.5 (1) 

0.5 (5) 

125-21226 13 1.0 - 

pBR322 DNA/BsuRI (HaeIII) Marker, 5 SM0271 0.5 50 100 0.5 (1) 8-587 22 2.5 

X174 DNA /BsuRI (HaeIII) Marker, 9 

X174 DNA /BsuRI (HaeIII) Marker, 9, 

ready-to-use 

SM0251 

SM0252 

SM0253 

0.5 

0.1 

50 

250 (5x50) 

50 

100 

500 

100 

0.5 (1) 

0.5 (5) 

72-1353 11 1.7 

5.0 

pUC19 DNA/MspI (HpaII) Marker, 23 

pUC19 DNA/MspI (HpaII) Marker, 23, 

ready-to-use 

SM0221 

SM0222 

SM0223 

0.5 

0.1 

50 

250 (5x50) 

50 

100 

500 

100 

0.5 (1) 

0.5 (5) 

26-501 13 1.7 5.0 

PAGE, 

% 




(for ready-to-use markers) 


0.005% bromophenol blue, 0.005% xylene 

cyanol FF and 10% glycerol. 



blue, 0.03% xylene cyanol FF, 60% glycerol and 

60 mM EDTA. 

Storage 


Ready-to-use versions can be stored at room 

temperature or at 4°C for 6 months.

Lambda DNA/EcoRI Marker, 1 

#SM0281 



21226* 218.8 43.8 

7421 76.5 15.3 

5804 59.8 12.0 

5643 58.2 11.6 

4878 50.3 10.1 

3530* 36.4 7.3 

0.5 μg/lane, 20 cm length gel, 1X TAE buffer, 3 V/cm, 

18 h (until bromophenol blue dye reached the bottom 

of the gel) 

Lambda DNA/HindIII Marker, 2 

#SM0101/2/3 



23130* 238.4 47.7 

9416 97.1 19.4 

6557 67.6 13.5 

4361* 45.0 9.0 

2322 23.9 4.8 

2027 20.9 4.2 

564 5.8 1.2 

125 1.3 0.3 



Lambda DNA/EcoRI+HindIII Marker, 3 

#SM0191/2/3 



21226* 218.8 43.8 

5148 53.1 10.6 

4973 51.3 10.3 

4268 44.0 8.8 

3530* 36.4 7.3 

2027 

1904 

20.9 

19.6 

4.2 

3.9 

1584 16.3 3.3 

1375 14.2 2.8 

947 9.8 1.95 

831 8.6 1.7 

564 5.8 1.2 



125 bp fragment is not visible and it comprises 0.3%.** 

* cohesive ends (the 12 nt cos site of lambda DNA) may anneal and form additional bands. These fragments can be separated by h eating at 65°C for 5 min and then cooling on ice for 3 min. 

** the shortest fragments (oblique) are not visible with standard electrophoresis methods. Fragment lengths are calculated using the respective DNA sequence (see p.357). 

pBR322 DNA/BsuRI (HaeIII) Marker, 5 

#SM0271 



587 67.3 13.5 

540 61.9 12.4 

502 57.6 11.5 

458 52.5 10.5 

434 49.8 10.0 

267 

234 

213 

192 

184 

124 

123 

104 

89 

30.6 

26.8 

24.4 

22.0 

21.1 

14.2 

14.1 

11.9 

10.2 

6.1 

5.4 

4.9 

4.4 

4.2 

2.8 

2.8 

2.4 

2.0 

80 9.2 1.8 


1X TBE, 5 V/cm, 1.5 h 


bp 

587* 

540* 

502 

458* 

434 

267 

234 

213 

192 

184 

124 

123 

104 

89 

80 

64 

57 

51 



X174 DNA/BsuRI (HaeIII) Marker, 9 

#SM0251/2/3 



1353 125.6 25.1 

1078 100.1 20.0 

872 81.0 16.2 

603 56.0 11.2 

310 

281 

271 

234 

194 

28.8 

26.1 

25.2 

21.7 

18.0 

5.8 

5.2 

5.0 

4.3 

3.6 

118 11.0 2.2 

72 6.7 1.3 


1X TBE, 5 V/cm, 1.5 h 


bp 

1353 

1078 

872 

603 

310* 

281* 

271* 

234 

194 

118 

72 



pUC19 DNA/MspI (HpaII) Marker, 23 

#SM0221/2/3 

21, 18, 11, 8 bp fragments are not visible and comprise 5.4%.** 

26 bp fragment is not visible and comprises 1.0%.** 

* bands form an anomalous pattern on polyacrylamide gels. 

** the shortest fragments (oblique) are not visible using standard electrophoresis methods. Fragment lengths are calculated using the respective DNA sequence (see p.357). 


bp ng/0.5μg % 

501 93.3 18.7 

489 

404 

91.0 

75.2 

18.2 

15.0 

331 61.6 12.3 

242 45.0 9.0 

190 

147 

111 

110 

67 

35.4 

27.4 

20.7 

20.5 

12.5 

7.1 

5.5 

4.1 

4.1 

2.5 

34 6.5 6.3 

34 6.5 6.3 

0.5μg/lane, 8cm length gel, 

1X TBE, 5V/cm, 1.5h 


b p 

501* 

489* 

404 

331 

242 

190 

147 

111 

110 

67 

34, 34 

0.5μg/lane, 20cm length gel, 

1X TAE, 8V/cm, 3h 


370 

NoLimits Individual DNA Fragments and Custom DNA Ladders 

DNA fragment 

size 

Cat. # 

Amount, 

μg 

10 bp SM1391 10 

15 bp SM1381 10 

20 bp SM1401 10 

25 bp SM1761 10 

35 bp SM1411 10 

50 bp SM1421 10 

75 bp SM1431 10 

100 bp SM1441 10 

150 bp SM1601 10 

200 bp SM1611 10 

250 bp SM1451 10 

300 bp SM1621 10 

400 bp SM1631 10 

500 bp SM1641 10 

600 bp SM1461 10 

700 bp SM1651 10 

750 bp SM1471 10 

800 bp SM1481 10 

850 bp SM1661 10 

900 bp SM1491 10 

1000 bp SM1671 10 

1200 bp SM1681 10 

1500 bp SM1691 10 

2000 bp SM1701 10 

2500 bp SM1571 10 

3000 bp SM1711 10 

3500 bp SM1501 10 

4000 bp SM1721 10 

5000 bp SM1731 10 

6000 bp SM1511 10 

7000 bp SM1741 10 

8000 bp SM1521 10 

10000 bp SM1751 10 

15000 bp SM1531 10 

17000 bp SM1771 10 

20000 bp SM1541 10 


Collection of Individual DNA Fragments 

Description 

The Thermo Scientific NoLimits Collection 

includes 36 h ighly purified individual DNA 

fragments ranging from 10 to 20,000 bp. 

NoLimits DNA fragments are produced using 

specifically designed plasmids purified through 

a proprietary technology. Plasmid DNA is 

digested with Thermo Scientific restriction 

enzymes and the individual DNA fragments are 

chromatography-purified from the digestion 

mixture. As a result, NoLimits fragments are 

exceptionally pure and free of any truncated 

or degraded molecules. These custom DNA 

fragments are the best choice for applications 

ranging from electrophoresis (both gel and 

capillary) to HPLC and beyond. 

1.1% TopVision Agarose (#R0491), 1X TAE, 7 V/cm, 30 min. 

NoLimits DNA fragments from 300 bp to 10 kb. 

NoLimits Custom DNA Ladders Service 

Custom DNA ladder formulations supplied in 

bulk quantities to fit your needs: 

Individual DNA fragments or mixtures. 

Custom ladders of any combination and 

concentration. 

Reference bands can be designed-in. 

Flexible formulations. 

Ready-to-use versions premixed with any 

loading dye (see p.372). 

From microgram to gram quantities. 

Features 

Chromatography-purified individual DNA 

fragments. 

Broad range – 10-20,000 bp. 

Sharp peaks during capillary electrophoresis; 

sharp bands after gel electrophoresis. 

Precise DNA concentration. 

Applications 

Gel electrophoresis. 

Capillary electrophoresis. 

DNA quantification. 

HPLC analysis. 

Applications where a custom DNA size 

standard is required. 

Concentration 

0.5 μg/μl 


10 mM Tris-HCl (pH 7.6) with 1 mM EDTA. 

Storage 


How to Order? 

Contact our local representative: 

see Contacts on 



Agarose Cat. # Size Applications Page 






Agarase p.319 

DNA Markers/Ladders pp.358-370 



Silica Bead DNA Gel Extraction Kit p.318 

Electrophoresis Buffers 

R0491 100 g 

 

R0492 500 g 

 

 

 

427 

427 






Buffer Cat. # Size 1X composition Application and features Usage recommendations 


(Tris-acetate-EDTA) 


(Tris-borate-EDTA) 


B49 1 L 

B52 1 L 




40 mM Tris 

20 mM acetic acid 

1 mM EDTA 

pH of 50X TAE: 8.4 

89 mM Tris 

89 mM boric acid 

2 mM EDTA 

pH of 10X TBE: 8.3 

Electrophoresis of nucleic acids in agarose and 

polyacrylamide gels. 

Used both as a running buffer and as a gel 

preparation buffer. 

Recommended for resolution of RNA and DNA 

fragments larger than 1500 b(p), for genomic 

DNA and for large supercoiled DNA. 

Filtered through a 0.22 μm membrane. 

Electrophoresis of nucleic acids in agarose and 




Recommended for electrophoresis of RNA and 

DNA fragments smaller than 1500 b(p). 


Storage 

There are no time limitations for storage of the 

electrophoresis buffers at room temperature. 

If the buffer is stored at lower temperatures, a 

precipitate may form, which is easily dissolved 

by gentle h eating. 

 

the electrophoresis run. 

 

50X TAE buffer to 980 ml of deionized 

water and mix well. 

 

capacity, therefore periodic replacement 

of the buffer during prolonged 

electrophoresis is recommended. 

 

the electrophoresis run. 

 

10X TBE buffer to 900 ml of deionized 

water and mix well. 

 

molecules migrate about 10% slower in 

TBE buffer than in TAE buffer. 








372 

Loading Dyes 












Description 

Thermo Scientific DNA Loading dye solutions 

are used to prepare DNA markers and 

DNA samples for loading on agarose or 

polyacrylamide gels. The optimized solutions 

contain different dyes (bromophenol blue, 

xylene cyanol FF or orange G) for visual tracking 

of DNA migration during electrophoresis. The 

presence of glycerol ensures that the DNA 

forms a layer at the bottom of the well. EDTA 

included in the solutions binds divalent metal 

ions and inhibits metal-dependent nucleases. 

6X DNA Loading Dye & SDS Solution is 

recommended for agarose gel analysis of DNA 

samples that contain h igh amounts of DNA 

binding proteins. The presence of SDS eliminates 

DNA-protein interactions and prevents gel-shifts. 

2X RNA Loading Dye is mainly used for 

preparation of RNA samples for electrophoresis, 

but is also used for denaturing DNA 

electrophoresis since it contains the denaturing 

agent formamide. 

100 kb 

10 kb 

1 kb 

100 bp 

10 bp 

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 

Agarose, % 

Tracking dye migration in agarose gels. 

Storage 



Bromphenol blue in TBE buffer 

Bromphenol blue in TAE buffer 

Xylene cyanol FF in TBE buffer 

Xylene cyanol FF in TAE buffer

Loading dye Cat. # Size, 

ml 

6X DNA 

Loading Dye 

6X MassRuler 

DNA Loading 

Dye 

6X Orange 

DNA Loading 

Dye 

6X TriTrack 

DNA Loading 

Dye 

6X DNA 

Loading Dye & 

SDS Solution 

2X RNA 

Loading Dye 

R0611 5x1 

R0621 5x1 

R0631 5x1 

R1161 5x1 

R1151 5x1 

R0641 1 

Composition Features Applications 

6X Solution 

 

bromophenol blue 

xylene cyanol FF 

 

 

adjusted with NaOH) 

6X Solution 

 


 

 


6X Solution 

 

orange G 


 

 


6X Solution 

 



orange G 

 

 


6X Solution 



 

 

 

adjusted with Tris) 

2X Solution 

 

 



 

 

 

DNA migration during 


 

with UV visualization of 


 

dependent nucleases. 

 



 



 


 



 



 


 



 



 


 

DNA-protein interactions, 

prevents appearance 

of additional bands due 

to annealing of DNA 

molecules with cohesive 

ends. 

 


 

DNA and RNA fragment 

migration during 


 


DNA or RNA fragments. 

 

denaturation DNA and 

RNA fragments. 

 

DNA for loading 

on agarose or 


 

DNA molecules. 

 


on agarose or 


 

DNA molecules. 

 


on agarose or 


 


on agarose or 


 

DNA samples 

containing h igh 

amounts of DNA 

binding proteins. 

 

 

gel analysis 

following restriction 

digestion, ligation or 

dephosphorylation 

reactions. 

 

DNA for loading on 

denaturing gels 

 

RNA for loading 

on agarose or 


* for more detailed information regarding the migration rates of dyes in agarose and polyacrylamide gels see Table 7.1 and Table 7.2 on p.374. 

Migration of dyes 

(1% agarose, 

TAE or TBE buffers) 

Xylene cyanol FF: 

TAE: 4160 bp 

TBE: 3030 bp 

Bromophenol blue: 

TAE: 370 bp 

TBE: 220 bp 

Bromophenol blue: 

TAE: 370 bp 

TBE: 220 bp 

Xylene cyanol FF: 

TAE: 4160 bp 

TBE: 3030 bp 

Orange G: 

TAE/TBE:

374 


7.1. General recommendations 

for DNA electrophoresis 

 

according to the tables below: 

Table 7.1. Recommended agarose gel percentages 

for electrophoretic separation of DNA fragments. 

Agarose 

gel, 

% 

Range of 

efficient 

separation, bp 


Approximate positions of tracking 

dyes, bp* 

Bromophenol blue Xylene cyanol FF 

TBE 

buffer 

TAE 

buffer 

TBE 

buffer 

TAE 

buffer 

0.5 2000-50000 750 1150 13000 16700 

0.6 1000-20000 540 850 8820 11600 

0.7 800-12000 410 660 6400 8500 

0.8 800-10000 320 530 4830 6500 

0.9 600-10000 260 440 3770 5140 

1.0 400-8000 220 370 3030 4160 

1.2 300-7000 160 275 2070 2890 

1.5 200-3000 110 190 1300 1840 

2.0 100-2000 65 120 710 1040 

3.0 25-1000 30 60 300 460 

4.0 10-500 18 40 170 260 

5.0 10-300 12 27 105 165 

* Positions of the tracking dyes can only be estimated approximately 

because the dye front migrates as wide band. 

Table 7.2. Recommended polyacrylamide gel 

percentages for electrophoretic separation of DNA 

fragments (1). 

Polyacrylamide gel 

(with BIS at 1:20), 

% (w/v) 

Range of 

efficient 

separation 

Approximate positions of 

tracking dyes* 

Bromophenol 

blue 

Xylene 

cyanol FF 

4.0 

Denaturing gels 

100-500 b 50 b 230 b 

5.0 70-400 b 35 b 130 b 

6.0 40-300 b 26 b 105 b 

8.0 30-200 b 19 b 75 b 

10.0 20-100 b 12 b 55 b 

15.0 10-50 b 10 b 40 b 

20.0 5-30 b 8 b 28 b 

30.0 1-10 b 6 b 20 b 

Non-denaturing gels 

3.5 100-1000 bp 100 bp 460 bp 

5.0 80-500 bp 65 bp 260 bp 

8.0 60-400 bp 45 bp 160 bp 

12.0 50-200 bp 20 bp 70 bp 

15.0 25-150 bp 15 bp 60 bp 

20.0 5-100 bp 12 bp 45 bp 

* Tracking dye positions can only be estimated as the dye front 

migrates as wide band. 

 

DNA fragments of various sizes: 

Size of the DNA Voltage Buffer 

< 1 kb 5-10 V/cm TBE 

1-5 kb 4-10 V/cm TAE or TBE 

> 5 kb 

Up to 10 kb, fast 

1-3 V/cm TAE 

electrophoresis with 

Express DNA ladders 

up to 23 V/cm TAE 

 

the DNA ladder/marker) for both sample DNA 

and ladder/marker DNA. 

 

DNA and the ladder/marker DNA. The sample 

can be diluted with 1X DNA loading dye. 

 

samples as this may cause band shift during 


 

gel staining in 0.5 μg/ml ethidium bromide 

solution, SYBR Green I or GelRed. 

7.1.1. Recommendations for accurate 

in-gel DNA quantification 

 

NTPs or buffer components can interfere with 

spectrophotometrical measurements and may 

lead to inaccurate quantification of sample 

DNA. In these cases, it is best to rely on gel 

quantification data. 

 

video-densitometry analysis. 

 

(supplied with the DNA ladder/marker) for 

both the sample DNA and the 

ladder/marker DNA. 

 

ladder band of the closest size. 

 

sample to approximately equalize it with the 

amount of DNA in the nearest band. 

7.2. Preparation of agarose gels 

for DNA electrophoresis 

7.2.1. Non-denaturing agarose gel 


Use a flask of at least three times greater 

volume than that of the solution to avoid 

boiling over. 

 


 

TBE Buffer (#B52) to a 1X concentration 

immediately before use. 

 

smaller than 1500 bp. For larger DNA, use 

TAE buffer. 

 

bromide to both the gel and the 

electrophoresis buffer at a final 0.5 μg/ml 

concentration. Alternatively, stain the gel 

after electrophoresis. 

Wear gloves when h andling ethidium bromide. 

 

plasmid DNA, ethidium bromide should not 

be included in the electrophoresis buffer 

or gel. The gel should be stained only after 

electrophoresis is complete. 

 

to UV light may cause DNA alterations. 

Therefore, minimize UV exposure to a few 

seconds if the purified DNA fragments will be 

used for cloning. 

Staining before electrophoresis with such 

intercalating dyes as SYBR GreenI, GelRed 

and others dyes may cause abberant 

migration of DNA bands and DNA Ladder, 

that may cause mistakes in sizing of DNA. 

Perform DNA staining step only after gel 


Preparation: 

1. Weigh out the required amount of agarose 

(#R0491 or #R0801) (depending on the gel 

percentage) into an Erlenmeyer flask. 

2. Add the appropriate volume of either 1X TBE 

or 1X TAE buffer and swirl to mix. 

3. Weigh the flask with the solution. 

For h igh percentage gels (3-5%): Add an 

excess amount of distilled water to increase the 

weight by 10-20%. 

4. Boil the mixture in a microwave oven (at 

medium power) until the agarose melts 

completely; swirl the flask several times 

while boiling. To prepare the h ighest 

quality agarose gels of any percentage, 

an additional 3-5 min of boiling after 

completely melting the agarose is 

recommended. A significant amount of 

water evaporates during this procedure 

and therefore restoring of the initial weight 

(in step 5) is required to obtain the desired 

percentage gel. 

5. Weigh the flask again and if necessary, add h 

ot distilled water to restore the initial weight. 

For h igh percentage gels (3-5%): Check (by 

weighing) that the excess 10-20% of water h as 

evaporated and, if needed, continue boiling to 

remove any excess, or add h ot distilled water to 

restore the initial weight. 

Optional: Add ethidium bromide to a final 

concentration of 0.5 μg/ml. Mix well and h eat for 

1 min without boiling. 


on a clean casting plate with an appropriate 

comb. If bubbles appear, push them 

carefully away to the sides with a pipette tip. 

7. Solidify the gel for approximately 30 min 

before use. Low percentage low melting point 

agarose gels need to be solidified at 4°C. 


electrophoresis buffer. Load the samples 

onto the gel. 

9. Run electrophoresis at 5-7 V/cm until the 

bromophenol blue runs approximately 2/3 of 

the way down the gel. 

10. After electrophoresis the gel can be stained 

by immersing it into a 0.5 μg/ml ethidium 

bromide solution for 15-20 min, stained 

with GelRed, SYBR Green I or any other DNA 

staining technique. 

Warning. Flasks containing h ot agarose 

solution should be h andled with caution.

7.2.2. Alkaline agarose gel 


Double stranded DNA ladders are not 

recommended for denaturing gel analysis 

as they may form an atypical pattern 

during electrophoresis. However, these 

discrepancies are normally acceptable for 

analysis of cDNA or other ssDNA in alkaline 

gels. 

 

volume than that of the solution to avoid a 

boil over event. 

 


(depending on the gel percentage) into an 

Erlenmeyer flask. 

2. Add the appropriate volume of the buffer 

(30 mM NaCl, 2 mM EDTA, pH 7.5) and 

swirl to mix. 

3-7. Follow steps from protocol 9.2.1. 

8. Immerse the gel for at least one h our 

into the alkaline electrophoresis buffer 

(30 mM NaOH, 2 mM EDTA). 

9. Load the samples. 

10. Run electrophoresis at 3 V/cm in alkaline 

electrophoresis buffer (30 mM NaOH, 2 mM 

EDTA) until the dye runs approximately 2/3 

of the way down the gel. 

After electrophoresis the gel should be 

immersed for 30 min in 100-300 ml of 

0.5 M Tris-HCl buffer, pH 7.5 and then 

stained in a 0.5 μg/ml ethidium bromide 

solution for 30 min. If staining is not 

sufficient, the whole procedure can be 

repeated. 

Warning. Hot agarose solution should be h 

andled very carefully. 

7.3. Preparation of gels for PAGE 

7.3.1. Non-denaturing PAGE 

Reagents: 

 

 

 

 

 

 

 

sample and ladder DNA 

1. For a nondenaturing 5% polyacrylamide gel 

solution of 40 ml, mix the following: 

10X TBE Buffer (#B52) 4 ml 

20% acrylamide/bisacrylamide 10 ml 

deionized water 26 ml 

Caution: Acrylamide is a neurotoxin; always 

wear gloves, safety glasses, and a surgical 

mask when working with acrylamide powder. 

2. Vigorously agitate the solution for 1 min by 

magnetic stirring to ensure complete mixing. 

3. Add 48 μl of TEMED and swirl the flask to 

ensure that the solution is thoroughly mixed. 

4. Immediately add 240 μl of fresh 10% (w/v) 

APS and mix thoroughly. 

5. Pour the acrylamide between the gel plates 

and insert the comb. 

Clamp the comb in place at the top of the 

gel to avoid separation of the gel from the 

plates as the acrylamide polymerizes. Allow 

the gel to polymerize for 30 min. 

Note 

Polymerization begins as soon as APS is added to the mixture, 

so all subsequent steps must be performed quickly. 

6. Remove the comb and any bottom spacers 

from the gel. Wash the gel plates to clean 

any spilled acrylamide and be sure that the 

spacers are properly seated and clean. Fill the 

lower reservoir of the electrophoresis tank with 

1X TBE buffer. Initially, place the gel into the 

lower tank at an angle to avoid the formation 

of air bubbles between the plates and the gel 

bottom. Clamp the gel plates to the top of the 

electrophoresis tank and fill the upper reservoir 

with 1X TBE so that the wells are covered. 

7. Pre-run and warm the gel for at least 

30 min at 5 V/cm (constant voltage). 

8. Prepare the ladders for electrophoresis as 

recommended in the product insert or in 

Table 7.3 on p.377. 

Load the recommended volume of the 

ladder, premixed with the appropriate 

loading dye solution. Use the same loading 

dye for the sample DNA. 

9. Run the gel at 5 V/cm, taking care to avoid 

excessive h eating. Run the gel for the time 

indicated in the certificate of analysis of the 

ladder. 

10. Stain the gel in a 0.5 μg/ml ethidium 

bromide aqueous solution for about 30 min. 

Examine the gel under the UV light. 

7.3.2. Denaturing polyacrylamide/ 

urea gel electrophoresis 

Reagents: 

 

 

 

 

 

water 

 

 

1. Prepare denaturing 10% polyacrylamide gel 

solution of 40 ml, mix the following: 


20% acrylamide/bisacrylamide 10 ml 

UREA 

19.2 g (to 8 M final 


deionized water to 40 ml 

Caution: Acrylamide is a neurotoxin; always 

wear gloves, safety glasses, and a surgical 

mask when working with acrylamide powder. 

2. Vigorously agitate the solution by magnetic 

stirring to ensure complete mixing and 

solving of UREA powder. 

3. Add 40 μl TEMED and swirl the flask to 

ensure thorough mixing. 

4. Immediately add 400 μl of fresh 10% (w/v) 

APS and mix thoroughly. 

5. Pour the acrylamide between the gel plates 

and insert the comb. 

6. Clamp the comb in place at the top of the 

gel to avoid separation of the gel from the 

plates as the acrylamide polymerizes. Allow 

the gel to polymerize for 30 min. 

Note 

Polymerization begins as soon as APS is added to the mixture, 

so all succeeding actions must be performed promptly. 

Run the gel: 

1. After polymerization is complete, remove the 

comb and any bottom spacers from the gel. 

Fill the lower reservoir of the electrophoresis 

tank with 1X TBE buffer. Initially, place the 

gel into the lower tank at an angle to avoid 

the formation of air bubbles between the 

plates and the gel bottom. Clamp the gel 

plates to the top of the electrophoresis tank 

and fill the upper reservoir with 1X TBE so 

that the wells are covered. 

2. Pre-run and warm the gel for at least 

30 min at 5 V/cm (constant voltage). 

Note 

During the gel run h eat the gel buffer to 60-70ºC. 

3. Wash the wells with 1X TBE buffer to 

remove UREA and gel debris. 

4. Load the samples. 

5. Run the gel at 6 V/cm until the lower dye 

front migrates 80% through the gel. 

bp 

300 

200 

150 

100 

75 

50 

35 

25 

20 

15 

bp 

300 

200 

150 

100 

Migration of Thermo Scientific GeneRuler Ultra 

Low Range and Low Range DNA ladders under 

denaturing conditions. 

Ladders are prepared for electrophoresis using 

formamide containing 2X RNA Loading Dye (#R0641) 

and separated on 10% denaturing PAGE with 8 M urea. 

1 – GeneRuler Ultra Low Range DNA Ladder (#SM1211) 

2 – GeneRuler Low Range DNA Ladder (#SM1191) 

75 

50 

25 


376 

6. Soak the gel for about 15 min in 1X TBE to 

remove the urea prior to staining. 

7. Stain the gel in a 0.5 μg/ml ethidium 

bromide aqueous solution for about 30 min. 

8. Examine the gel under the UV light. 

Note 

 

pattern in denaturing electrophoresis. However, GeneRuler 

Ultra Low Range and Low Range DNA Ladders can be 

successfully used for sizing ssDNA (see Figure on p.375). 

Ultra Low Range DNA Ladder: 10 bp band is not 

visible, 15 bp is the first one seen. 

Low Range DNA Ladder: upper bands 

(400, 500, 700 bp) are not separated well under denaturing 

conditions. 

7.4. Preparation of DNA samples 

for electrophoresis 

7.4.1. Preparation of DNA samples for 

conventional DNA electrophoresis 

6X DNA Loading Dye (#R0611), 6X MassRuler 

DNA Loading Dye (#R0621), 6X Orange DNA 

Loading Dye (#R0631), 6X TriTrack DNA 

Loading Dye (#R1161) are all used according to 

below protocol: 

1. Add 1 volume of 6X DNA loading dye to 

5 volumes of DNA sample. 

2. Mix well, spin down and load. 

7.4.2. Preparation of DNA samples for 

denaturing polyacrylamide/urea gel 


Note 

Use the same loading dye solution for the sample and the 

ladder DNA. 

1. Mix the DNA sample with an equal volume 

of 2X RNA Loading Dye (#R0641). 

2. Heat at 95ºC for 5 min. 

3. Chill the sample on ice for 3 min. 

4. Keep samples on ice while loading. 

7.4.3. Preparation of DNA samples 

for denaturing alkaline agarose gel 


1. Mix 5 volumes of the DNA sample or ladder 

with one volume of 6X alkaline electrophoresis 

loading buffer (180 mM NaOH, 6 mM EDTA, 

18% Ficoll 400, 0,05% bromcresol green). 

2. Heat samples and ladder at 70°C for 5 min, 

then chill on ice for 3 min. Load onto the gel. 

7.4.4. Preparation of DNA samples 

containing DNA binding proteins 

Use 6X DNA Loading Dye & SDS Solution 

(#R1151) to prevent the appearance of 

additional bands or gel shifts when analyzing: 

– probes after DNA restriction digestions, 

ligation or dephosphorylation reactions, 

– DNA samples with h igh amounts of DNA 

binding proteins, 

– DNA molecules with cohesive ends, 

– or to stop an enzymatic reaction during 

kinetic experiments. 


1. Add 1 volume of 6X DNA Loading Dye & 

SDS Solution to 5 volumes of DNA sample. 

2. Mix well and h eat at 65°C for 10 minutes. 

3. Chill on ice, spin down and load. 

Note 

The prepared sample can be stored at -20ºC and reused for 

electrophoresis after several freeze-thaw cycles. 

M 1 2 3 4 5 6 7 

The effect of SDS on electrophoresis on resolving 

band shifts. 

M – GeneRuler DNA Ladder Mix (#SM0331) 

1 – 0.5 μg DNA prepared for loading with 6X DNA 

Loading Dye (#R0611) 

2 – 0.5 μg DNA prepared for loading with 6X DNA 

Loading Dye & SDS Solution (#R1151) 

3 – 0.5 μg DNA digested with FastDigest FokI 

(#FD2144), prepared for loading with 6X DNA 

Loading Dye 

4 – 0.5 μg DNA digested with FastDigest FokI, prepared 

for loading with 6X DNA Loading Dye & SDS Solution 

5 – 0.4 μg of the 2 DNA fragment ligation mixture 

prior to ligation 


after the ligation, prepared for loading with 



after the ligation, prepared for loading with 

6X DNA Loading Dye & SDS Solution 

7.5. Preparation of DNA ladders/markers for electrophoresis 

Recommendations for loading of DNA ladders/markers supplied in TE buffer. 

Technical specifications 

Amount used per 1 mm width of a 

gel lane 

Conventional formulation (supplied in TE buffer) DNA ladders/markers 

GeneRuler DNA ladders 

0.1 μg (0.2 μl) for agarose gel 

0.2 μg (0.4 μl) for PAGE 

Heating Do not h eat 

I. Loading on agarose gel: 


loading dye 

Water, nuclease-free (#R0581) 

II. Loading on polyacrylamide gel: 


loading dye 

Water, nuclease-free (#R0581) 

1 μl (0.5 μg) 

2 μl 

9 μl 

Mix gently and load on gel 

Conventional Lambda DNA 

markers 


Heat at 65°C for 5 min; 

chill on ice for 3 min before use 

1 μl (0.5 μg) 

2 μl 

9 μl 

2 μl (1 μg) 

0.5 μl 

Not recommended for PAGE 

0.5 μl 

Mix gently and load on gel 

Conventional Phage & 

Plasmid DNA markers 


0.2 μg (0.4 μl) for PAGE 

Do not h eat 

1 μl (0.5 μg) 

2 μl 

9 μl 

2 μl (1 μg) 

0.5 μl 

0.5 μl 

7.6. Labeling of DNA ladders/markers 

For protocols see chapter 6 Thermo Scientific Molecular Labeling & Detection Products on p.349.

7.7. Separation of Express DNA ladders in different electrophoresis conditions 

Table 7.3. Thermo Scientific GeneRuler Express DNA 

Ladder (#SM1551) fragment separation under various 


Duration of 0.8% 1% 1.2% 1.5% 2% 2.5% 

electropho- agarose agarose agarose agarose agarose agarose 

resis 

23 V/cm 

5 min 

10 min 

15 min 

20 min 

TAE TBE TAE TBE TAE TBE TAE TBE TAE TBE TAE TBE 

Table 7.4. Thermo Scientific MassRuler Express 

HR Forward and Reverse DNA Ladder (#SM1243 

and #SM1253) fragment separation under various 


Duration of 


7V/cm 

10 min 

20 min 

30 min 

40 min 

50 min 

0.7% 

agarose 

1% 

agarose 

1.3% 

agarose 

1.5% 

agarose 

TAE TBE TAE TBE TAE TBE TAE TBE 


LR Forward and Reverse DNA Ladder (#SM1283 



Duration of 

electropho- 

0.7% 

agarose 

1% 

agarose 

1.3% 

agarose 

1.5% 

agarose 

Reference 

1. Sambrook, J., et al., Molecular Cloning. A Laboratory 

Manual, Cold Spring Harbor Laboratory, Cold Spring 

Harbor, N.Y., 12.89, 5.42, 2001. 

2% 

agarose 

resis 

TAE TBE TAE TBE TAE TBE TAE TBE TAE TBE 

7 V/cm 

10 min 

20 min 

30 min 

40 min 

50 min 


Forward and Reverse DNA Ladder Mix (#SM1263 



Duration of 


7 V/cm 

10 min 

20 min 

30 min 

40 min 

50 min 

0.7% 

agarose 

1% 

agarose 

1.3% 

agarose 

1.5% 

agarose 

TAE TBE TAE TBE TAE TBE TAE TBE 

Table 7.7. Thermo Scientific ZipRuler Express DNA Ladder (#SM1373) fragment separation under various 


Duration 

0.8% agarose 1% agarose 1.2% agarose 1.5% agarose 1.7% agarose 2% agarose 

of electro- TAE TBE TAE TBE TAE TBE TAE TBE TAE TBE TAE TBE 

phoresis 

10 V/cm 

1 

5 min 

10 min 

15 min 

20 min 

25 min 

Ladder 

2 Mix 1 2 Mix 1 

Ladder 

2 Mix 1 2 Mix 1 

Ladder 

2 Mix 1 2 Mix 1 

Ladder 

2 Mix 1 2 Mix 1 

Ladder 

2 Mix 1 2 Mix 1 

Ladder 

2 Mix 1 2 Mix 

1 – ZipRuler Express DNA Ladder 1. 

2 – ZipRuler Express DNA Ladder 2. 

Mix – Ladder 1 and Ladder 2 mixed at equal volumes. 

Excellent separation of all bands. 

Incomplete separation of the two closest bands. 

No separation. 

Lowest bands run off of gel (8 cm). 


378 


1 

Low intensity 

of all or some 

DNA bands 

1.1 

Insufficient 

amount of 

DNA loaded 

1.2 

Insufficient 

or uneven 

staining 

1.3 

DNA 

run off of 

the gel 

1.4 

DNA 

diffusion in 

the gel 

1.5 

DNA masking 

by tracking 

dyes 


2 

Smeared DNA 

bands 

2.1 

DNA 

degradation 

by nucleases 

2.2 

Incorrect electrophoresis 

conditions 

2.3 

Gel shift 

effect 

2.4 

Excess 

DNA loaded 

2.5 

High salt 

concentration 

in samples 

2.6 

Poorly 

formed gel 

wells 

DNA electrophoresis 

problem 

3 

Atypical 

banding 

pattern 

3.1 

marker not h 

eated prior to 

loading 

3.2 

Denatured 

DNA 

3.3 

Different 

loading conditions 

for ladder 

& sample 

3.4 


conditions 

3.5 

DNA 

staining before 


3.6 

Atypical migration 

due to 

DNA sequence 

or structure 

3.7 

Gel shift 

effect 

3.8 

High salt 

concentration 

in samples 

4 

Curved DNA 

bands 

4.1 

Gel 

incompletely 

immersed in 

buffer 

4.2 

Low sample 

volume 

4.3 


conditions 

4.4 

Bubbles or 

physical particles 

in wells 

or in the gel 

5 

DNA remains 

in the gel well 

5.1 

Poorlyformed 

gel wells 

5.2 

Excess DNA 

loaded 

5.3 

Contamination 

of the DNA 

sample 

5.4 

Gel shift 

effect 

6 

Incorrect 


data 

6.1 

Different 


for ladder 

& sample 

6.2 

Incorrect 

ladder band 

chosen for 


6.3 

Improper 


method 

6.4 

Uneven or h 

igh backgroundstaining 

of the gel 

6.5 

DNA masking 

by tracking 

dyes

Table 7.8. Troubleshooting guide for DNA electrophoresis. 


1.1. Insufficient amount of ladder was loaded. 

Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers 

(~0.1-0.2 μg per 1 mm gel lane width). 

1.2. Insufficient or uneven staining. 

Following electrophoresis, visualize DNA by staining in ethidium bromide solution (final concentration 

0.5 μg/ml) or SYBR Green I. 

Alternatively, if the DNA will not be used for cloning, add ethidium bromide to both the gel and 

electrophoresis buffer at a final 0.5 μg/ml concentration. 

After alkaline agarose gel electrophoresis the gel should be immersed for 30 min in 300 ml 0.5 M Tris-HCl 

buffer, pH 7.5 and only later stained in a 0.5 μg/ml ethidium bromide solution for 30 min. 

After denaturing polyacrylamide gel electrophoresis with urea, soak the gel for about 15 min in 1X TBE to 

remove the urea prior to staining. Stain the gel in 0.5 μg/ml ethidium bromide in 1X TBE solution for 15 min. 

Make sure that the gel is immersed completely in the staining solution. 

1. Low intensity of all or some of 

the DNA bands 

2. Smeared DNA bands 

1.3. DNA run off the gel. 

Perform electrophoresis until the bromophenol blue dye passes 2/3 (orange G, 4/5) of the gel. Refer to the 

table on p.374 for migration of tracking dyes in different gels. 

Make sure that the entire gel is immersed completely in the electrophoresis buffer during the run. Make sure 

that the gel and apparatus are positioned h orizontally during the run. 

1.4. DNA diffusion. 

Avoid prolonged electrophoresis or excessive staining and destaining procedures as this may cause diffusion 

of smaller DNA fragments in the gel. 

Avoid long term storage of the gel before taking a picture, as this may cause diffusion of DNA fragments and 

low band intensity. 

1.5. DNA masking by electrophoresis tracking dyes. 

Do not exceed the amount of electrophoresis tracking dyes used for sample/ladder preparation. Use the 

loading dye solutions supplied with every Thermo Scientific DNA ladder/marker, as these solutions contain 

amount of tracking dyes that will not mask DNA under UV light. 

Prepare DNA ladders and probes according to recommendations on p.376. 

2.1. DNA degradation by nucleases. 

Use fresh electrophoresis buffers, freshly poured gels, nuclease-free vials and tips to minimize nuclease 

contamination of DNA solutions. 

2.2. Incorrect electrophoresis conditions. 

Prepare gels according to the recommendations on p.374, always use the same electrophoresis buffer for 

both the gel and running buffer. 

Ensure that the whole gel is immersed completely in the electrophoresis buffer during the run. 

Do not use an excessively h igh voltage for electrophoresis. Run the gels at 5-8 V/cm. To increase the band 

sharpness, use a lower voltage for several minutes at the beginning of electrophoresis. 

Excessively h igh voltage may result in gel h eating and DNA denaturation. 

For fast electrophoresis under h igh voltage (up to 23 V/cm) use GeneRuler or O’GeneRuler Express DNA 

ladders (#SM1551/2/3 or #SM1563, p.361). 

An excessively low voltage during the entire run may result in diffusion of bands during electrophoresis. 

To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value 

(usually 5-8 V/cm, depending on the ladder) one h as to: 

– measure the distance between electrodes (cathode and anode) – X, cm 

– and multiply that X, cm value by the recommended voltage (Y, V/cm) 

– the result (X, cm x recommended Y, V/cm) is Z – recommended voltage to be applied. 

2.3. Gel shift effect. 

DNA binding proteins, such as ligases, phosphatases or restriction enzymes may alter DNA migration on gels 

and cause the DNA to remain in the gel well or gel shifting. 

Lambda DNA or other DNA with long complementary overhangs may anneal and migrate atypically (see p.369). 

To correct for the above mentioned effects, use 6X DNA Loading Dye & SDS Solution (#R1151, p.373) 

which is supplemented with 1% SDS to eliminate DNA-protein interactions and to prevent annealing of DNA 

molecules via long cohesive ends. 

Always h eat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load. 

(continued on next page) 


380 



2. Smeared DNA bands 

3. Atypical banding pattern 


2.4. Excess DNA loaded. 

Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers 

(~0.1-0.2 μg per 1 mm gel lane width) or in the Table on p.376. Use similar DNA quantities for the samples. 

2.5. High salt concentration in the sample. 

Samples containing h igh concentrations of salts may result in smeared or shifted band patterns. 

Ethanol precipitation and washing the pellet with ice cold 75% ethanol or spin column purification prior to 

resuspending the sample in water or TE buffer h elps to eliminate salts present in the sample. 

2.6. Poorly formed (slanted) gel wells. 

When inserting the comb into the gel, make sure that it is vertical to the gel surface and stable during gel 

casting and solidification. 

3.1. Lambda DNA marker was not h eated prior to loading. 

All DNA markers generated from Lambda DNA, as well as lambda DNA digestion products, should be h 

eated at 65°C for 5 min and chilled on ice before loading on the gel in order to completely denature the 

cohesive ends (the 12 nt cos site of lambda DNA) that may anneal and form additional bands. See p.376 

for preparation of lambda markers for electrophoresis. 

3.2. Denatured DNA. 

Excessively h igh voltage may result in gel h eating and DNA denaturation. 

To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (often 

5-8 V/cm, depending on the ladder) one h as to: 


– and multiply that X, cm value by the recommended voltage (Y, V/cm) 

– the result (X, cm x recommended Y, V/cm) is Z – recommended voltage to be applied. 

For non-denaturing electrophoresis use the loading dye solutions supplied with every Thermo Scientific DNA 

ladder/marker, as these solutions do not contain denaturing agents. 

Prepare DNA ladders and probes according to recommendations on p.376. 

Do not h eat them before loading. Heating is required only for lambda DNA markers. 

3.3. Different loading conditions for sample and ladder DNA. 

Always use the same loading dye solution (supplied with the DNA ladder/marker) for both the sample DNA 

and the ladder/marker DNA. 

If possible, always load equal or very similar volumes of the sample DNA and the ladder/marker DNA. The 

sample can be diluted with 1X loading dye. 


Excessive electrophoresis run times or voltage may result in migration of small DNA fragments off of the gel. 

Very short or slow electrophoresis may result in incompletely resolved bands. 

Run gels at 5-8 V/cm until the bromophenol blue passes 2/3 (orange G, 4/5) of the gel. Refer to the Table 

7.1 on p.374 for migration of tracking dyes in different gels. 



TAE buffer is recommended for analysis of DNA fragments larger than 1500 bp and for supercoiled 

DNA. TBE buffer is used for DNA fragments smaller than 1500 bp and for denaturing polyacrylamide gel 

electrophoresis. Large DNA fragments will not separate well in TBE buffer. 

The correct gel percentage is important for optimal separation of the ladder DNA; prepare gels according 

to recommendations on p.374. When preparing agarose gels always adjust the volume of water to 

accommodate for evaporation during boiling. Otherwise, the gel percentage will be too h igh and result in 

bad separation of larger DNA bands. 

Refer to the Table 7.1 on p.374 for the range of effective separation of DNA in different gels. 





3.5. DNA staining before electrophoresis using fluorescent dyes. 

Ethidium bromide interferes with separation of large DNA fragments. Do not include ethidium bromide in the 

gel and run buffer when large DNA (more than 20 kb) or supercoiled DNA is analyzed. Stain the gel following 

electrophoresis in a 0.5 μg/ml ethidium bromide solution for 30 min. 

Staining before electrophoresis with intercalating dyes such as SYBR Green I, GelRed and others may cause 

abberant migration of DNA bands leading to errors in fragment size determination. Perform DNA staining 

following gel electrophoresis. 

3.6. Atypical migration due to differences in DNA sequence or structure. 

During h igh resolution electrophoresis, DNA fragments of equal size can migrate differently due to variations 

in DNA sequences. AT rich DNA may migrate slower than an equivalent size GC rich DNA fragment. The 

sequences of Thermo Scientific DNA ladders are chosen to allow for h ighly accurate DNA migration 

according to size, h owever, due to differences in nucleotide sequence or the overall DNA structure, sample 

migration can sometimes slightly differ from ladder band migration. 

DNA structures such as nicked, supercoiled or dimeric molecules will always show different mobility on gels 

compared to an equivalent DNA size standard. See the picture below for migration of plasmid DNA forms: 

bp 

10000 

8000 

6000 

5000 

4000 

3500 

3000 

2500 

2000 

1500 

1000 

750 

500 

250 

1 2 3 

1 GeneRuler 1 kb DNA Ladder (#SM0311) 

2 Undigested plasmid pUC19 2,7 kb DNA, forms: 

– upper band (~4 kb) – dimeric plasmid 

– below, less visible (~3.5 kb) – nicked plasmid 

– lowest band (~1.9 kb) – supercoiled plasmid. 

3 Linearized plasmid pUC19 (2,7 kb) – migrates according to its size 

High level DNA modifications such as methylation, labeling with biotin or large fluorescent molecules also 

result in slower migration compared to unmodified DNA of the same size. 


The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes 

may alter DNA migration in the gel or cause the DNA to remain in the gel wells. 

Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical 

migration pattern. 

To eliminate these effects, use 6X DNA Loading Dye & SDS Solution (#R1151) that is supplemented with 1% SDS 

to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends. 


High salt concentration in the sample may also cause gel shift effects, see below (3.8). 

3.8. High salt concentration in the sample. 

Samples with a h igh salt concentration may give smeared or shifted band patterns. 

Ethanol precipitation and washing the pellet with ice cold 75% ethanol or spin column purification prior 

resuspending DNA in water or TE buffer h elps eliminate salt from the sample. 



382 



4. Curved DNA bands 

5. DNA remains in the gel well 

6. Incorrect quantification data 


4.1. Gel incompletely immersed in electrophoresis buffer. 

Electrophoresis buffer should completely cover the entire gel during sample loading and run. 

4.2. Low sample volume. 

The sample or the ladder volume should be large enough to fill 1/3 of the total capacity of the well. Large 

wells should not be used with small sample volumes. If needed the sample volume can be adjusted with 

1X loading dye. 


Do not use an excessively h igh voltage for electrophoresis. Run the gels at 5-8 V/cm. To minimize band 

curving, use a lower voltage for several minutes at the beginning of electrophoresis. 



To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (which 

is in many cases 5-8 V/cm, depending on the ladder) one h as to: 


– and multiply the X value by the recommended voltage (Y, V/cm) 

– the result (X x Y) is the recommended voltage to be applied. 

4.4. Bubbles or physical particles in the gel wells or in the gel. 

Use pure water, clean flasks and clean equipment to prepare gels. 

Pour the gel slowly to avoid bubble. Bubbles can be removed with a pipette tip prior to gel solidification. 

5.1. Poorly formed gel wells. 

Remove the gel comb only after complete polymerization of the gel. Pour the buffer onto the gel immediately. 

Rinse the wells with electrophoresis buffer to remove urea from denaturing polyacrylamide gels prior to 

loading the sample. 

5.2. Excess DNA loaded. 

Follow the recommendations for loading described in the certificate of analysis supplied with the DNA ladders/ 

markers (~0.1-0.2 μg per 1 mm gel lane width) or in the Table 7.3 on p.377. If possible load the same quantity of 

the sample. 

5.3. Contamination of the DNA sample. 

Ensure that your sample DNA solution does not contain any precipitate. 


The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes 

may alter DNA migration in the gel and cause the DNA to remain in the gel wells. 

Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical band 

migration pattern. 

To eliminate these effects, use 6X DNA Loading Dye & SDS Solution that is supplemented with 1% SDS to 

eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends. 


6.1. Different loading conditions for sample and ladder DNA. 

Always use the same loading dye solution (supplied with the DNA ladder/marker) for both the sample DNA 

and the ladder/marker DNA. 

If necessary, adjust the concentration of the sample to approximately equalize it with the amount of DNA in 

the nearest band. 

Use equal or very similar volumes of the sample DNA and the ladder/marker DNA. The sample can be diluted 

with 1X loading dye solution. 

6.2. Incorrect ladder band chosen for sample quantification. 

Always compare the sample band with a ladder band of similar size. 

6.3. Improper quantification method used. 

If possible, quantify by video-densitometry while subtracting the gel background as this method is more 

precise than a visual comparison of the bands. 




6.4. Uneven staining of the gel or h igh background staining. 

Ensure that the gel is immersed completely in the staining solution. 

Following electrophoresis, visualize DNA by staining in ethidium bromide solution (final concentration 

0.5 μg/ml) or SYBR Green I. Do not exceed the recommended concentration of the dye for staining. 

Avoid prolonged staining for more than 30 min as this may result in h igh background. 

If the gel is to be stained during the run, ensure that the ethidium bromide is included in both the gel and 

running buffer, otherwise the staining will be uneven. 

After alkaline agarose gel electrophoresis the gel should be immersed for 30 min in 300 ml of 


0.5 M Tris-HCl buffer, pH 7.5 and only later stained in a 0.5 μg/ml ethidium bromide solution for 30 min. 

After denaturing polyacrylamide gel electrophoresis with urea, soak the gel for about 15 min in 1X TBE to 


6.5. DNA masking by electrophoresis tracking dyes. 

Do not exceed the recommended amount of electrophoresis tracking dyes used for sample/ladder 

preparation. Use the loading dye solutions supplied with every Thermo Scientific DNA ladder/marker, as 

these solutions contain equilibrated amount of tracking dyes that will not mask DNA under UV light. 

Prepare DNA ladders and probes according to the recommendations on p.376. 


384 



RNA Electrophoresis Products 

RNA Ladders .....................................................................................................................386 

RiboRuler RNA Ladders (100-6000 bases) ..................................................................386 

RiboRuler Low Range RNA Ladder .............................................................................386 

RiboRuler High Range RNA Ladder .............................................................................386 

Double-stranded RNA Analysis .................................................................................... 387 

GeneRuler Ultra Low Range DNA Ladders.................................................................. 387 

Phi6 dsRNA ...................................................................................................................387 

Reagents for RNA Electrophoresis ..............................................................................388 

2X RNA Loading Dye ....................................................................................................388 

Electrophoresis Buffers ..............................................................................................389 

Agarose .........................................................................................................................389 

Protocols and Recommendations ...............................................................................390 

8.1. General recommendations for RNA electrophoresis ....................................................390 

8.2. Preparation of RNA ladders for electrophoresis ..........................................................390 

8.3. Preparation of RNA samples for electrophoresis ........................................................390 

8.4. Preparation of gels for RNA electrophoresis ...............................................................390 

Troubleshooting Guide ...................................................................................................392 

Find out more about 

RNA Electrophoresis 

products online: 

www.thermoscientific.com/ladders 


386 

RNA Ladders 

RiboRuler RNA Ladders (100-6000 bases) 

RNA ladder Cat. # 


2X RNA Loading Dye 1 ml 


GeneJET RNA Purification Kit p.307 

GeneJET Plant RNA Purification Kit 

GeneJET Whole Blood RNA Purification 

p.309 

Mini Kit p.308 





Agarase p.319 


Products for RNA Synthesis in vitro 

TranscriptAid T7 High Yield in vitro 

p.325 

Transcription Kit p.328 


#SM1821 

1.0% native TopVision Agarose (#R0491) 

2 μl of SM1821/lane, 


1X TAE, 5 V/cm 

bases ng/2 μl 

6000 120 

4000 120 

3000 120 

2000 120 

1500 120 

1000 120 

500 120 

200 120 


» 6.1.3. Radiolabeling of RNA ladders 


» 8.1. General recommendations for 

RNA electrophoresis p.390 

» 8.2-8.3. Preparation of RNA 

ladders/samples for electrophoresis p.390 

» 8.4. Preparation of gels for RNA 



1.0% formaldehyde agarose 

bases 

6000 

4000 

3000 

2000 

1500 

1000 

500 

200 

2 μl of #SM1821/lane, 


1X MOPS, 5 V/cm 

Description 

Thermo Scientific RiboRuler RNA ladders are 

composed of chromatography-purified singlestranded 

RNA transcripts and are free from 

NTPs and RNA degradation products. 

The RiboRuler High Range RNA Ladder is a 

mixture of 8 single-stranded RNA transcripts 

ranging in size from 200 to 6000 b. 

The RiboRuler Low Range RNA Ladder is a 

mixture of 7 single-stranded RNA transcripts 

ranging in size from 100 to 1000 b. The ladders 

are supplied in conventional format premixed 

with 1 mM EDTA, pH 6.0 or in a ready-to-use 

format, premixed with RNA loading dye. 

RiboRuler RNA ladders supplied in 

conventional format can be end-labeled using 

T4 Polynucleotide Kinase (#EK0031), and are 

ideal for use in Northern blot applications. 


#SM1831 

2% native TopVision Agarose (#R0491) 

Volume, 

μl 

bases ng/2 μl 

1000 

800 

140 

140 

600 140 

400 140 

300 140 

200 140 

100 140 



1X TAE, 5 V/cm 

Applications 

5.0% polyacrylamide-urea 

bases 

1000 

800 

600 

400 

300 

200 

100 



1X TBE, 8 V/cm 

Loading, 

μl/4-8 mm 

lane 

RiboRuler Low Range RNA Ladder 

RiboRuler Low Range RNA Ladder, ready-to-use 

SM1831 

SM1833 

100 (5x20) 

200 (5x40) 

50-100 

1-2 

2-4 

RiboRuler High Range RNA Ladder 

RiboRuler High Range RNA Ladder, ready-to-use 

SM1821 

SM1823 

100 (5x20) 

200 (5x40) 

50-100 

1-2 

2-4 

Range, 

bases 

Number of 

fragments 

Agarose, 

% 

PAGE, 

% 

100-1000 7 1.7-2.5 4.0-8.0 

200-6000 8 0.8-1.5 – 

Features 

Sharp bands of uniform intensity. 

Easy-to-remember band sizes and quantities. 

Ideal for in-gel RNA quantification. 

Convenient – available in both conventional 

and ready-to-use formats. 

Supplied with 2X RNA Loading Dye. 

Stable for 6 months at -20°C. 

Applications 

RNA sizing and quantification on native or 

denaturing gels. 

Northern blotting. 


1 mM EDTA (pH 6.0). 


(for ready-to-use ladders) 

47.5% formamide, 0.0125% SDS, 

0.0125% bromophenol blue, 

0.0125% xylene cyanol FF, 

0.0125% ethidium bromide and 

0.75 mM EDTA. 

2X RNA Loading Dye 

95% formamide, 0.025% SDS, 

0.025% bromophenol blue, 

0.025% xylene cyanol FF, 

0.025% ethidium bromide and 0.5 mM EDTA. 

Storage 

Store at -70°C (or at -20°C for 6 months). 

Fluorescence 

20 

15 

10 

5 

0 

1500 b 

20 25 30 35 40 45 50 55 60 65 

Time (seconds) 

Analysis of Thermo Scientific RiboRuler 

High Range RNA Ladder (#SM1821) using an 

Agilent 2100 bioanalyzer.

Double-stranded RNA Analysis 

GeneRuler Ultra Low Range DNA Ladders 

DNA ladders for 

small RNA analysis 


DNA Ladder 



O’GeneRuler Ultra Low Range 


bp 

300 

200 

150 

100 

75 

50 

35 

25 

20 

15 

10 

Cat. # 


μg/μl 

Amount, 

μg 

Applications, 

0.5 g/lane 

SM1211 

50 50-100 

0.5 

SM1212 250 (5x50) 250-500 

1 2 

siRNA gel analysis using Thermo Scientific GeneRuler or 

O’GeneRuler Ultra Low Range DNA ladders. 

Electrophoresis conditions: 5% agarose gel, 1X TBE buffer, 7 V/cm, 45 min. 

1 – GeneRuler Ultra Low Range DNA Ladder. 

2 – 21 bp siRNA (0.5 μg), premixed with 6X DNA Loading Dye (#R0611). 

Phi6 dsRNA 

21 bp siRNA 

#F-630 20 μg (0.5 μg/μl) 


Phi6 RNA Replicase p.332 


NTP Set p.411 

M – HindIII Digest. 

1 – The Phi6 RNA fragments 

2948 bp, 4063 bp and 6374 bp. 

Loading, 

μg(μl)/ 

lane 

0.5-1 (1-2) 

SM1213 0.1 50 100 0.5-1 (5-10) 

SM1223 0.1 50 100 0.5-1 (5-10) 

Description 

Thermo Scientific GeneRuler Ultra Low 

Range DNA Ladders are composed of 11 

chromatography-purified individual fragments. 

The ladders are available in TE buffer or in two 

ready-to-use formats, premixed with different 

loading dyes. 

The presence of very small DNA fragments in 

the GeneRuler Ultra Low Range DNA ladder 

allow for siRNA gel analysis. 

Description 

Thermo Scientific Phi6 dsRNA ladder is an 

equimolar mixture of three linear doublestranded 

RNA fragments segments that 

constitute the bacteriophage Phi6 genome. 

The RNA fragments are 2948 bp, 4063 bp 

and 6374 bp in length. The relative fragment 

concentrations are approximately 110, 150 

and 240 ng/μl, respectively. 

Range, 

bp 

Number of 

fragments 

Application 

small dsRNA analysis. 

Agarose, 

% 

PAGE, 

% 

Page 

10-300 11 4.5-5.0 8-10 359 

Applications 

dsRNA size standard for agarose gel 


Control RNA for dsRNA-specific enzymatic 

reactions; e.g., Dicer, RNaseIII or 

RNA helicases. 

Source 

The dsRNA is isolated from the wild-type 

bacteriophage Phi6 propagated in 

P. syringae pv. phaseolicola HB10Y. 


dsRNA mixture is supplied in: 

10 mM Tris-HCl (pH 8.0), 1 mM EDTA. 

Unit definition 

1.0 A260 unit dsRNA = 40 μg/ml. 


388 

Reagents for RNA Electrophoresis 

2X RNA Loading Dye 

#R0641 1 ml 






Agarase p.319 

Products for RNA synthesis in vitro p.325 






ladders/samples for electrophoresis p.390 




Description 

2X RNA Loading Dye is recommended for 

preparation of RiboRuler RNA ladders and 

RNA samples for electrophoresis on agarose 

or polyacrylamide gels. It contains the tracking 

dyes bromophenol blue and xylene cyanol FF, as 

well as the intercalating dye ethidium bromide. 

In most denaturing agarose gel systems 

bromophenol blue migrates slightly faster than 

human 5S rRNA, whereas xylene cyanol FF 

migrates slightly slower than 18S rRNA. 

The 2X RNA Loading Dye contains the 

denaturing agent formamide which allows RNA 

fragments to separate according to size even 

during non-denaturing electrophoresis and acts 

as an RNA stabilizer. 

xylene 

cyanol FF 

bromophenol 

blue 

Migration of tracking dyes. 

Application 

Preparation of RNA for loading on agarose or 


Composition of 2X Solution 

 

 

 

 

 

 

Storage 

Store at -20°C (or at 4°C for up to 12 months).

Electrophoresis Buffers 

Buffer Cat. # Size 1X composition Application and features Usage recommendations 


(Tris-acetate-EDTA) 


(Tris-borate-EDTA) 





Agarase p.319 

Agarose 

Storage 

The electrophoresis buffers can be stored 

indefinitely at room temperature. If the buffer 

is stored at lower temperatures, a precipitate 

may form, which is easily dissolved by gentle 

heating. 

Agarose Cat. # Size Applications Page 


B49 1 L 

B52 1 L 

40 mM Tris 

20 mM acetic acid 

1 mM EDTA 

pH of 50X TAE: 8.4 

89 mM Tris 

89 mM boric acid 

2 mM EDTA 

pH of 10X TBE: 8.3 


Electrophoresis of nucleic acids in agarose 

and polyacrylamide gels. 



Recommended for resolution of RNA and DNA 

fragments larger than 1500 b(p), for genomic 

DNA and for large supercoiled DNA. 


 


 


 

DNA fragments smaller than 1500 b(p). 

 

R0491 100 g 

 

R0492 500 g 

 

 

 

 

electrophoresis run. 

 

buffer to 980 ml of deionized water and mix well. 

 

capacity; therefore, periodic replacement of 

the buffer during prolonged electrophoresis is 

recommended. 

 

electrophoresis run. 

 

buffer to 900 ml of deionized water and mix well. 

 

migrate about 10% slower in TBE buffer than in 

TAE buffer. 

427 

427 





ladders/samples for RNA electrophoresis p.390 




390 


8.1. General recommendations 

for RNA electrophoresis 

RNA is extremely sensitive to degradation 

by ribonucleases. Therefore, use only fresh 

electrophoresis buffers and freshly 

poured gels. 

 

RNA gel analysis avoid electrophoresis tanks 

used for DNA miniprep analysis since DNA 

minipreps may contain RNase A or T1. 

 

samples and for RNA markers. 2X RNA 

Loading Dye is provided with all RiboRuler 

RNA ladders and 2X RNA Loading Dye is 

available separately (#R0641). It contains 

ethidium bromide for RNA visualization 

on denaturing formaldehyde gels. If RNA 

is separated on native agarose gels or on 

polyacrylamide/urea gels, additional staining 

with ethidium bromide is recommended. 

 

bromide to both the agarose gel and running 

buffer. 

8.2. Preparation of RNA ladders 


For RiboRuler RNA ladders (#SM1831 and 

#SM1821): 

1. Mix 1 volume of RNA ladder and 1 volume of 

the supplied 2X RNA Loading Dye (#R0641). 

2. Heat at 70°C for 10 min. 

3. Chill on ice for 3 min and spin down prior to 

loading on a gel. 

4. Load 0.5 μl of the prepared ladder for 

every mm of gel lane width (4 μl / 8 mm lane). 

For RiboRuler RNA ladders, ready-touse 

(#SM1833 and #SM1823): 

1. Heat RNA ladders at 70°C for 10 min. 

2. Chill on ice for 3 minutes and spin down 

prior to loading on a gel. 

3. Load 0.5 μl of the ladder for every mm of 

gel lane width (4 μl / 8 mm lane). 

Note 

Ladders prepared as described above are not suitable for 

glyoxal/DMSO agarose gel electrophoresis. To prepare ladders 

for glyoxal/DMSO agarose gels, refer to the protocol 10.4.3. 

8.3. Preparation of RNA samples 


1. Mix 1 volume of the 2X RNA Loading 

Dye (#R0641) and 1 volume of the RNA 

sample. 

2. Heat at 70ºC for 10 min. 

3. Chill on ice for 3 minutes and spin down 

prior to loading on a gel. 


Note 

RNA samples prepared as described above are not suitable 

for glyoxal/DMSO agarose gel electrophoresis. To prepare 

RNA samples for glyoxal/DMSO agarose gels, refer to the 

protocol 10.4.3. 

8.4. Preparation of gels for RNA 


8.4.1. Non-denaturing agarose gels 

 

times greater volume than that of the 

solution to avoid boiling over. 

 


 

buffers to a 1X concentration immediately 

before use. 

 

smaller than 1500 b. For larger RNA 

fragments, use TAE buffer. 


(depending on the gel %) into an Erlenmeyer 

 

2. Add the appropriate volume of either 1X TBE 

or 1X TAE buffer and swirl to mix. 

3. 

For high percentage gels (3-5%): add an 

excess amount of distilled water to increase the 

weight by 10-20%. 

4. Boil the mixture in a microwave oven (at 

middle power) until the agarose melts 

 

while boiling. To prepare the highest 

quality agarose gels of any percentage, 

an additional 3-5 min of boiling after 

completely melting the agarose is 

recommended. A significant amount of 

water evaporates during this procedure 

and, therefore, restoring the initial weight 

(in step 5) is required to obtain the desired 

percentage gel. 

5. 

hot distilled water to restore the initial weight. 

For high percentage gels (3-5%): check (by 

weighing) that the excess 10-20% of water has 

evaporated and, if needed, continue boiling to 

remove any excess, or add hot distilled water to 

restore the initial weight. 

Optional. Add ethidium bromide to a final 

concentration of 0.5 μg/ml. Mix well and heat 

for 1 minute without boiling. 


into a clean casting tray. Remove bubbles 

with a pipette tip. 

7. Allow the gel to solidify for approximately 

30 min before use. Low percentage low 

melting point agarose gels can be solidified 

at 4°C. 


electrophoresis buffer. 

9. Heat the RNA samples and ladder at 70°C 

for 10 min, then chill on ice for 3 min. Load 

onto the gel. 

10. Perform electrophoresis at 5 V/cm until the 



After electrophoresis, the gel can be stained by 

immersing it into a 0.5 μg/ml ethidium bromide 

solution for 20 min, stained with SYBR Green II 

or any other RNA staining technique. 

Warning. Hot agarose solution should be 

handled very carefully. 

8.4.2. Denaturing formaldehyde gels in 

MOPS buffer 

1. Prepare fresh 10X MOPS buffer: 

– 0.4 M MOPS (pH 7.0), 

– 0.1 M sodium acetate, 

– 0.01 M EDTA (pH 8.0). 

2. Prepare 1% TopVision Agarose (#R0491) gel: 

– stir 1g of agarose powder in 72 ml of 

deionized water, 

– melt the agarose, and then add 10 ml of 

10X MOPS buffer and mix, 

– cool to 60°C and add 18 ml of fresh 

37% formaldehyde (in a fume hood) and 

mix thoroughly, 

– pour the gel. 

3. Place the gel into an electrophoresis 

apparatus containing 1X MOPS buffer. 

4. Heat the RNA samples and ladder at 70°C 

for 10 min, and then chill on ice for 3 min. 

5. Load onto the gel. 

Note 

There is no need to stain the gel as the ethidium bromide 

present in 2X RNA Loading Dye is sufficient for visualization 

under UV light. 

8.4.3. Denaturing glyoxal/DMSO gels 

in sodium phosphate buffer 

1. Prepare a thick 1% TopVision 

Agarose (#R0491) gel in 0.01 M sodium 

phosphate buffer, pH 7.0. 

2. Place the gel into an electrophoresis 

apparatus with 0.01 M sodium phosphate 

buffer, pH 7.0. 

3. For loading prepare 25 μl aliquots of the 

ladder/samples by adding: 

Glyoxal (40% solution) 4.5 μl 

DMSO 12.5 μl 

0.1 M sodium phosphate buffer, pH 7.0 2.5 μl 

4. Mix and add: 

RiboRuler RNA Ladder or RNA sample 3 μl 

2X RNA Loading Dye 1 μl 


5. Incubate for 1 hour at 50°C and then cool 

down to room temperature.

6. Load the samples on a gel. 

7. Run electrophoresis at 5 V/cm until the 



8. Stain the gel in 0.5 μg/ml ethidium bromide 

solution in 0.5 M ammonium acetate for 

15-30 min. 

9. Wash the gel in fresh 0.5 M ammonium 

acetate solution for 15-30 min. 

8.4.4. Denaturing polyacrylamide/ 

urea gels in TBE buffer 

Prepare 20 ml of a 5% polyacrylamide gel 

containing 7 M urea by adding: 

47.5% acrylamide: 2.5% bis-acrylamide 

solution 

2 ml 

10 M urea 14 ml 


10% freshly prepared ammonium persulfate 0.2 ml 

Deionized water 1.8 ml 

1. Mix and add 10 μl of TEMED. Mix again and 

pour the gel carefully avoiding formation of 

air bubbles. 

2. Insert the comb into the acrylamide and allow 

the gel to polymerize for at least 1 hour. 

3. Fill the electrophoresis apparatus with 

1X TBE buffer. 

4. Heat RNA samples and ladder at 70°C for 

10 min and chill on ice for 3 min. 

5. Load the samples on a gel. 

6. Run electrophoresis at 8 V/cm for about 

1 hour. 

7. Soak the gel for about 15 minutes in 1X TBE 

to remove urea prior to staining. 

8. Stain the gel in 0.5 μg/ml ethidium bromide 

in 1X TBE solution for 15 min. 


392 


1 

RNA bands 

not visible 

1.1 

Insufficient 

staining 

1.2 

No staining 

1.3 

Insufficient 

amount of 

ladder loaded 

1.4 

RNA 

degradation 

1.5 

RNA 

diffusion 

1.6 

RNA 

run off 

the gel 


2 

Smeared RNA 

bands 

2.1 

RNA 

degradation 

2.2 

Improper 

ladder storage 

or usage 

2.3 

Gel depth 

inhibits RNA 

mobility 

2.4 

Improper electrophoresis 

conditions 

2.5 

Excess ladder 

loaded 

2.6 

Incompletely 

immersed 

gel 

3 

Atypical RNA 

ladder 

banding pattern 

RNA electrophoresis 

problem 

3.1 

Inefficient denaturation 

of 

the ladder 

3.2 

Sub-optimal 

gel 

preparation 

3.3 

Different 


for ladder 

& sample 

3.4 

Improper electrophoresis 

conditions 

3.5 

Sub-optimal 

ethidium bromide 

conc. 

3.6 

Incompletely 

immersed 

gel 

4 

High 

background 

staining 

4.1 

Excess ethidium 

bromide/ 

excess 

staining 

4.2 

Insufficient 

destaining 

5 

Uneven 

staining of 

the gel 

5.1 

Improper gel 

staining 

conditions 

5.2 

Incompletely 

immersed 

gel 

6 

RNA remains 

in the gel well 

6.1 

Poorly formed 

gel wells 

6.2 

Large quantity 

RNA loaded on 

the gel 

6.3 

Contamination 

of the RNA 

sample 

7 

Incorrect 


data 

7.1 

Impure RNA 

7.2 

Incorrect 

ladder band 

chosen 

7.3 

Different 


for ladder 

and sample 

7.4 

Improper 


method 

7.5 

Uneven or high 

background 

staining of the 

gel

Table 8.1. Troubleshooting guide for RNA electrophoresis. 


1. RNA bands are not visible 

2. Smeared RNA bands 

1.1. Insufficient staining. 

Use the 2X RNA Loading Dye to prepare the conventional RiboRuler RNA ladder and RNA sample prior to 

electrophoresis. The loading dye solution includes ethidium bromide at a concentration sufficient to stain 

RNA on denaturing formaldehyde agarose gels. Ready-to-use RiboRuler RNA ladders are premixed with 

2X RNA Loading Dye. 

If RNA fragments will be separated on native agarose gels, additional staining of the gel with ethidium 

bromide (final concentration 0.5 μg/ml) is recommended. 

If RNA is separated on a denaturing glyoxal/DMSO agarose gel, stain the gel in ethidium bromide solution 

(final concentration 0.5 μg/ml) in 0.5 M ammonium acetate for 15-30 min after electrophoresis. Wash the 

gel in a fresh 0.5 M ammonium acetate solution for 15-30 min. 

If RNA is separated on a denaturing polyacrylamide gel with urea, soak the gel for about 15 min in 1X TBE to 


1.2. No staining. 

If you are using a loading dye which does not contain ethidium bromide, add ethidium bromide to both the 

agarose gel and electrophoresis buffer at a final concentration of 0.5 μg/ml. 

Alternatively, stain the gel after electrophoresis with ethidium bromide (0.5 μg/ml ethidium bromide) for 

20 min, or SYBR Green II (follow supplier recommendations). 

1.3. Insufficient amount of ladder was loaded. 

Follow the recommendations for loading described in the certificate of analysis supplied with each of the 

RiboRuler RNA ladders (0.25 μl per mm gel lane for conventional ladders; 0.5 μl per mm gel lane for readyto-use 

ladders). 

1.4. RNA degradation. 

Minimize exposure to UV light as this may cause RNA degradation/fading. 

RNA is extremely sensitive to degradation by ribonucleases. The use of fresh electrophoresis buffers, freshly 

poured gels, DEPC-treated solutions and protective gloves is recommended. 

1.5. RNA diffusion. 

Avoid prolonged electrophoresis or excessive staining and destaining procedures as this may cause diffusion 

of smaller RNA fragments from the gel. 

Avoid long term storage of the gel prior to photo documentation, as this may cause diffusion of RNA 

fragments and band fading. 

1.6. RNA run off the gel. 

Stop electrophoresis after the bromophenol blue passes 2/3 of the length of the gel. In most denaturing 

agarose gel systems, bromophenol blue migrates slightly faster than 5S rRNA and xylene cyanol FF migrates 

slightly slower than 18S rRNA. 

Make sure that the electrophoresis tank is in a completely vertical position. 

2.1. RNA degradation. 

RNA is extremely sensitive to degradation by ribonucleases. The use of fresh electrophoresis buffers, freshly 

poured gels, DEPC-treated solutions and protective gloves is recommended. 

2.2. Improper storage or use of RNA ladders. 

Store RiboRuler RNA ladders at -20°C for 6 months or at -70°C for 24 months. Thaw the ladders on ice. 

2.3. Excessive gel depth or sample volume. 

Use thin (~0.5 cm) gels and avoid loading of large volumes in the gel lane. 

2.4. Improper electrophoresis conditions. 

Ensure that there is enough electrophoresis buffer in the electrophoresis apparatus and that the gel is 

immersed completely. 

Do not use an excessively high voltage for electrophoresis. Run agarose gels at 5 V/cm (polyacrylamide/urea 

gels at 8 V/cm). To increase the band sharpness, use a lower voltage for several minutes at the beginning of 


However, very low voltage during the entire run may result in band diffusion. 

2.5. Excessive RNA ladder loaded onto the gel. 

Follow the recommendations for loading described in the certificate of analysis of the RiboRuler RNA ladders 

(0.25 μl per mm gel lane for conventional ladders; 0.5 μl per mm gel lane for ready-to-use ladders). 

2.6. Incompletely immersed gel. 

Always ensure that there is enough electrophoresis buffer in the electrophoresis apparatus. 



394 




4. High background staining 


3.1. In-sufficient ladder denaturation. 

All RiboRuler 

centrifuged before loading on the gel in order to completely denature the RNA. Sample RNA should be 

prepared the same way. 

3.2. Sub-optimal gel preparation. 

Older formaldehyde has an acidic pH which may cause extra RNA bands to form on the gel. Use only fresh 

formaldehyde for optimal results. 

3.3. Different loading conditions for the sample and the ladder. 

Both ladder and sample RNA should be prepared with the same loading dye solution and loaded under the 

same conditions. 

After electrophoresis of total RNA samples in the presence of ethidium bromide, the 28S and 18S human 

rRNA should be clearly visible under UV illumination. Fast-migrating bands composed of 5.8S RNA and 5S 

RNA may also be visible depending on the RNA purification procedure. The intensity of the 28S RNA should 

be approximately twice the intensity of the 18S RNA. 

The 28S human rRNA band migrates at approximately 5000 b and the 18S human rRNA band migrates at 

approximately 1900 b. 

3.4. Improper electrophoresis conditions. 

Excessively long electrophoresis runs may result in migration of small RNA fragments off the gel. 

Very short electrophoresis runs may result in incompletely resolved bands. 

Run agarose gels at 5 V/cm (polyacrylamide/urea gels at 8 V/cm) until the bromophenol blue passes 2/3 of 

the gel length. 

TAE buffer is recommended for analysis of larger RNA, and TBE buffer is used to resolve RNA fragments 

smaller than 1500 b and for denaturing polyacrylamide gel electrophoresis. 

The correct gel percentage is important for optimal separation of the ladder RNA. 

Optimal conditions for RiboRuler High Range RNA Ladder (#SM1821/3): 

– native 0.8-1.5% agarose gel with TAE buffer, 

– denaturing formaldehyde 0.8-1.5% agarose gel with MOPS buffer, 

– denaturing glyoxal/DMSO 0.8-1.5% agarose gel with sodium phosphate buffer. 

Optimal conditions for RiboRuler Low Range RNA Ladder (#SM1831/3): 

– native 1.7-2.5% agarose gel with TBE buffer, 

– denaturing formaldehyde 1.7-2.5% agarose gel with MOPS buffer, 

– denaturing glyoxal/DMSO 1.7-2.5% agarose gel with sodium phosphate buffer, 

– denaturing 4-8% polyacrylamide gel with TBE buffer. 

3.5. Sub-optimal ethidium bromide concentration in sample and ladder. 

The 2X RNA Loading Dye allows for RNA visualization without the need for additional staining of denaturing 

agarose gels. Adding extra ethidium bromide to the ladder or sample is not recommended and may result in 

RNA migration toward the cathode. 

If RNA fragments are separated on native agarose gels or on polyacrylamide/urea gels, additional staining 

with ethidium bromide after electrophoresis is recommended. 


Always ensure that there is enough electrophoresis buffer in the electrophoresis apparatus. 

4.1. Excessively high ethidium bromide concentration or prolonged staining. 

Use ethidium bromide at a final concentration of 0.5 μg/ml. 

Avoid prolonged gel staining. 

4.2. Insufficient gel destaining. 

If the gel is extensively stained with ethidium bromide, additional destaining in water is needed to 

remove the background staining. 

Wash glyoxal/DMSO agarose gels after staining in a fresh 0.5 M ammonium acetate solution for 15-30 min. 




5. Uneven gel staining 

6. RNA remains in the gel well 


5.1. Improper gel staining conditions. 

Ethidium bromide migrates in the opposite direction of the RNA during electrophoresis. Therefore, if ethidium 

bromide is only added to the agarose gel and not to the electrophoresis buffer, it may result in uneven RNA 

fragment staining. 

When 2X RNA Loading Dye is used for both conventional RiboRuler RNA ladders and RNA sample 

preparation prior to electrophoresis, additional staining is not required as the loading dye includes sufficient 

ethidium bromide to stain RNA on denaturing formaldehyde agarose gels. 

For native agarose gels, ethidium bromide (0.5 μg/ml) should be added to both the electrophoresis buffer 

and the agarose. This ensures an even distribution of ethidium bromide during electrophoresis so that the 

intensity of the bands upon exposure to UV light will be proportional to the quantity of RNA present. 


Always ensure that there is enough electrophoresis buffer in the electrophoresis apparatus or enough of the 

staining solution during the staining so that the gel is always immersed completely. 

6.1. Poorly formed gel wells. 

Remove the gel comb only after complete polymerization of the gel. Pour the buffer onto the gel immediately. 

Rinse the wells with electrophoresis buffer to remove urea from denaturing polyacrylamide gels prior to 

loading the sample. 

6.2. Large quantity of RNA loaded on the gel. 

Follow the recommendations for loading described in the certificate of analysis of the RiboRuler RNA ladders 

(0.25 μl per mm gel lane for conventional ladders; 0.5 μl per mm gel lane for ready-to-use ladders). 

6.3. Contamination of the RNA sample. 

Ensure that your sample RNA solution does not contain any precipitate. 

7.1. Impure RNA. 

Free NTPs and truncated transcripts remaining in the sample after in vitro transcription can interfere with 

spectrophotometrical measurements and lead to inaccurate quantification of sample RNA. 

RiboRuler RNA ladders are produced from chromatography-purified RNA transcripts and are free 

of any NTPs and truncated transcripts. Therefore, the gel quantification data is compatible with the 

spectrophotometrical measurements of RiboRuler RNA ladders. 

7.2. Incorrect RiboRuler band chosen for quantification of the sample. 

Always compare the sample band to a ladder band of similar size. 

7.3. Different loading conditions for the ladder and samples. 

Both sample and ladder RNA should be loaded under the same conditions. 

Use the supplied 2X RNA Loading Dye for the sample and ladder. 

Load equal volumes of sample RNA and ladder RNA. The required volume of sample RNA can be obtained by 

dilution with a mixture (1:1) of DEPC-treated Water (#R0603) and 2X RNA Loading Dye. 

7.4. Improper quantification method used. 

If possible, quantify by video-densitometry measurements while subtracting the gel background as this 

method is more precise than a visual comparison of the bands. 

7.5. Uneven staining of the gel and high background staining can also interfere with gel quantification 

results (see Problem 4 and 5 above). 


396 



Epigenetics Products 

Practical Solutions for Epigenetics .............................................................................398 

EpiJET DNA Methylation Analysis Kit (MspI/HpaII) .....................................................399 

EpiJET DNA Methylation Analysis Kit (TaqI/HpyF30I) .................................................399 

EpiJET 5-hmC Analysis Kit ..........................................................................................400 

CpG Methyltransferase (M.SssI) ..................................................................................401 

SgeI ...............................................................................................................................402 

CpG Methylated Human Genomic DNA .......................................................................403 

CpG Methylated Jurkat Genomic DNA ........................................................................403 

Jurkat Genomic DNA ....................................................................................................403 


398 

Practical Solutions for Epigenetics 

Discover new Thermo Scientific tools designed 

for fast, efficient and robust epigenetic 

analysis. Our product line contains kits that 

utilize methylation-discriminating restriction 

enzymes as well as other modifying enzymes 

that aid in the analysis of 5-methylcytosine and 

5-hydroxymethylcytosine modifications. 

5-mC analysis 

5-mC 

EpiJET DNA Methylation Detection Analysis Kit 

(MspI/HpaII). 

EpiJET DNA Methylation Analysis Kit 

(TaqI/HpyF30I). 

CpG Methyltransferase (M.SssI). 

SgeI Restriction Enzyme. 


Fast epigenetic analysis. 

Reduced overall reaction times without 

compromising reaction efficiency. 

Specially formulated, highly stable enzymes 

and reagents for epigenetics. 

Tools for Epigenetic 

Analysis 

Controls 

CpG Methylated Human Genomic DNA. 

CpG Methylated Jurkat Genomic DNA. 

Jurkat Genomic DNA. 

5-hmC analysis 

EpiJET 5-hmC Analysis Kit.

EpiJET DNA Methylation Analysis Kit (MspI/HpaII) 




GeneJET Whole Blood DNA Purification Kit p.312 

CpG Methylated Human Genomic DNA p.403 

CpG Methylated Jurkat Genomic DNA p.403 

Jurkat Genomic DNA p.403 


(TaqI/HpyF30I) p.399 

Description 

5-Methylcytosine is a prominent epigenetic DNA 

modification which plays an important role in 

the regulation of gene expression. 

The Thermo Scientific EpiJET DNA Methylation 

Analysis Kit (MspI/HpaII) uses Epi MspI and 

Epi HpaII restriction enzymes to analyze DNA 

methylation status at a specific locus. Epi MspI 

and Epi HpaII are isoschizomers with different 

sensitivities to CpG methylation. When the CpG 

in the 5’-CCGG-3’ tetranucleotide sequence is 

EpiJET DNA Methylation Analysis Kit (TaqI/HpyF30I) 








 

(MspI/HpaII) p.399 

Description 

5-Methylcytosine is a prominent epigenetic 

DNA modification which plays an important role 

in up- and down-regulation of gene expression. 

In mammalian cells 5-methylcytosines are 

mainly found in 5’-CpG-3’ dinucleotides and 

are enriched in CpG islands, the GC-rich regions 

most often located in gene promoters. 

MspI/HpaII enzyme pair with 5’-CCGG-3’ 

recognition sequence has multiple targets in 

such regions and is routinely used for cytosine 

methylation analysis. Outside the CpG islands the 

CCGG motives are much less abundant and limit 

the usability of the aforementioned enzyme pair. 

The Thermo Scientific EpiJET DNA Methylation 

methylated, cleavage with Epi HpaII is blocked, 

but cleavage with Epi MspI is not affected. 

The Epi MspI and Epi HpaII enzymes are 

specially formulated for epigenetics studies to 

complete genomic DNA digestion in 1 hour. 

Features 

Fast – complete digestion of genomic DNA 

in 1 hour at 37°C. 

Efficient – specially formulated enzymes for 

epigenetic studies of DNA methylation status. 

Applications 

CpG methylation analysis at 5’-CCGG-3’ loci. 


 

 

 

Control pUC19/SmaI DNA Unmethylated 

(0.5 μg/μl) 

Control pUC19/SmaI DNA CpG Methylated 

(0.5 μg/μl) 

Storage 


Analysis Kit (TaqI/HpyF30I) uses another pair 

of restriction enzymes to analyze the DNA 

methylation status in gene bodies and other 

low-GC content genomic regions. HpyF30I and 

TaqI are isoschizomers with TCGA specificity; 

when cytosine in the 5’-TCGA-3’ tetranucleotide 

is methylated, the cleavage with Epi HpyF30I is 

blocked, but cleavage with Epi TaqI is not affected. 

The Epi HpyF30I and Epi TaqI enzymes are 

specially formulated for the epigenetic studies 

to complete genomic DNA digestion in 1 hour. 

Features 

Unique – novel tool for DNA methylation 

analysis in 5’ TCGA 3’ sequences. 

Efficient – enzymes specifically formulated 

for genomic DNA methylation analysis. 

Robust – reproducible data, correlating with 

other analysis methods. 

Fast procedure – Complete digestion of 

genomic DNA in 1h. 

Applications 

CpG methylation analysis at the 5’-TCGA-3’ 

loci. 


 

 

 

1 2 3 4 5 6 

Parallel digestion of methylated 

and unmethylated DNA. 

Methylated and unmethylated linearized plasmid DNA 

(pUC19/SmaI) were mixed with human blood genomic 

DNA and digested in parallel with methylation-sensitive 

Epi HpaII restriction enzyme and methylation insensitive 

isoschizomer Epi MspI for 1 hour at 37°C. Methylated 

plasmid DNA and naturally methylated gDNA were 

protected from cleavage with Epi HpaII but were cleaved 

with methylation-insensitive Epi MspI. Unmethylated 

plasmid DNA was cleaved with both enzymes. 

1, 4 – undigested DNA (control) 

2, 5 – DNA following digestion with Epi HpaII 

3, 6 – DNA following digestion with Epi MspI 

 

(0.5 μg/μl) 

 

(0.5 μg/μl) 

Storage 


pUC19 DNA / SmaI mpUC19 DNA / SmaI 

genomic DNA 

plasmid DNA 

1 2 3 4 5 6 

Digestion of methylated and unmethylated DNA 

by Epi HpyF30I and Epi TaqI. 

1, 4 – undigested DNA, 

2, 5 – digested with Epi TaqI, 

3, 6 – digested with Epi HpyF30I. 

1-3 contain unmethylated pUC19/SmaI plasmid. 

4-6 methylated with CpG Methyltransferase (M.SssI). 

Genomic DNA in all lanes is human blood gDNA. 


400 

EpiJET 5-hmC Analysis Kit 








Glucosylation 

Restriction digestion 

qPCR 

15 min. 

4 hours 

~ 2 hours 

5-hmC detection workflow. m-C methylcytosine, hm-C 

hydroxymethylcytosine, ghm-C glucosylated hydroxymethylcytosine. 


Genomic DNA 

m hm 

 

5'....CCGG....3' 5'....CCGG....3' 5'....CCGG....3' 

3'....GGCC....5' 3'....GGCC....5' 3'....GGCC....5' 

 

m hm 

+ T4 – glucosyltransferase + UDP-glucose 

m ghm 

 

5'....CCGG....3' 5'....CCGG....3' 5'....CCGG....3' 

3'....GGCC....5' 3'....GGCC....5' 3'....GGCC....5' 

 

m ghm 

+ Epi MspI 

m ghm 

 

5'' 5'' 5'....CCGG....3' 

3'' 3'' 3'....GGCC....5' 

 

m ghm 

m ghm 

 

5'' 5'' 5'....CCGG....3' 

3'' 3'' 3'....GGCC....5' 

 

m ghm 

Description 

The Thermo Scientific EpiJET 5-hmC Analysis 

Kit is an efficient system for detection of 

5-hydroxymethylcytosine (5-hmC) within specific 

DNA loci. The kit takes advantage of the ability 

of the T4 phage -glucosyltransferase (T4 BGT) 

to add a glucose moiety to 5-hmC residues, 

as well as the sensitivity of restriction enzyme 

MspI to glucosylated 5-hmC. The EpiJET 

5-hmC Analysis Kit includes two advanced 

enzymes: T4 BGT and Epi MspI. The T4 BGT 

is formulated for highly specific, complete and 

fast glucosylation of 5-hmC in 15 min. The Epi 

MspI is an MspI restriction enzyme specially 

formulated for efficient use in epigenetic studies. 

It completely digests genomic DNA in 4 hours 

when cytosine, 5-methylcytosine, or 5-hmC is 

within its recognition sequence, but its activity is 

completely blocked when 5-hmC is glucosylated. 

The assay involves DNA sample glucosylation 

and subsequent Epi MspI digestion. Restriction 

digestion products are then analyzed by 

qPCR with a primer pair flanking the CCGG 

site of interest. Results are compared against 

non-gluosylated and non-digested controls. 

A 

B 

C 

D 

Cq1 < Cq2 < Cq3 

Features 

Complete and specific 5-hmC conversion to 

glucosylated 5-hmC in 15 min. 

Discrimination of internal 5-hmC in CCGG 

sequences by Epi MspI digestion in 4 hours. 

Quantification of internal 5-hmC content in 

CCGG sequences by qPCR. 

Applications 

Internal 5-hmC analysis and quantification in 

CCGG sequences. 


T4 – glucosyltransferase 

Epi Buffer 

UDP–glucose 

Epi MspI 

5-hmC Control DNA 

Control primer 1 

Control primer 2 

Water, nuclease-free. 

Note 

Modification of the external C in CCGG 

sequences blocks the activity of Epi MspI. 

This may affect the digestion efficiency of 

non-glucosylated DNA. Levels of external C 

methylation vary depending on sample type 

and origin. 

Cq1 Cq2* 

Cq3 - Cq1 > 7 cycles 

Full hydroxymethylation. 

Glucosylation blocks cleavage by Epi 

MspI. CCGG site is fully 5-hydroxymethylated. 

Cq1 < Cq2 < Cq3 

Partial hydroxymethylation. 

Cleavage level represents the hydroxymethylation 

level in the CCGG site of 

interest. 

Cq3 Cq2* 

Cq2 - Cq1> 7 cycles 

No hydroxymethylation. CCGG 

site does not contain 5-hmC bases. 

Glucosylation does not affect cleavage 

by Epi MspI. 

Cq1 Cq2 Cq3* 

No cleavage. No Epi MspI recognition 

site or CCGG sequence modified 

at external C (Epi MspI cleavage is 

blocked). 

Interpretation of results. 

Cq1 – threshold cycle of “-T4 BGT-Epi MspI” sample. Cq2 – threshold cycle of 

“+T4 BGT+Epi MspI” sample. Cq3 – threshold cycle of “-T4 BGT+Epi MspI” sample 

* stands for Cq 0.5

CpG Methyltransferase (M.SssI) 

#EM0821 25 μl 


 

 



GeneJET Whole Blood DNA Purification Kit 

EpiJET DNA Methylation 

p.312 

Analysis Kit (MspI/HpaII) 


p.399 







vendor A vendor B 

15 min at 37°C 2 h at 30°C 1 h at 37°C 

1 2 3 1 2 3 1 2 3 

Plasmid DNA methylated for 15 min using Thermo Scientific M.SssI is 

protected from HpaII cleavage. 0.5 μg of linearized plasmid DNA (pUC19/ 

SmaI) was methylated using M.SssI from different suppliers according to vendor 

recommendations. The methylated plasmid DNA was mixed with 0.5 μg of human 

blood genomic DNA and digested in parallel with the methylation-sensitive HpaII 

restriction enzyme and methylation insensitive isoschizomer MspI. Methylated 

plasmid DNA and naturally methylated gDNA were protected from cleavage by HpaII 

and cleaved by MspI. 

1 – undigested DNA (control) 

2 – after digestion with HpaII 

3 – after digestion with MspI 

Description 

The Thermo Scientific CpG Methyltransferase 

(M.SssI) is one of the basic tools used in 

epigenetic studies. The enzyme methylates the 

C 5 position on the base moiety of all cytosine 

nucleotides contained in unmethylated or 

hemimethylated double stranded DNA in a 

5’-CpG-3’ context. 

Thermo Scientific M.SssI is specifically 

formulated for fast methylation times without 

compromising reaction efficiency. The enzyme 

completes modification of all CpGs in 15 min 

at 37°C. In addition, the enzyme has been 

specifically validated for use with genomic 

DNA – the primary substrate in epigenetic 

studies. 

 

 

(SAM) as a cofactor. 

genomic DNA 

plasmid DNA 

Features 

High efficiency – complete in vitro 

methylation of all CpG dinucleotides in 

non-methylated and hemi-methylated DNA. 

Fast – reaction is completed in 15 min at 

37°C. 

Stable – at -20°C for 2 years. 

Applications 

Epigenetics studies. 

In vitro methylation of DNA for methylation 

analysis. 

Inhibition of endonucleases with overlapping 

CpG sequence recognition. 

[ 3H]-labeling of DNA. 


CpG Methyltransferase (M.SssI) is supplied in: 

10 mM potassium phosphate (pH 7.0 at 25°C), 

400 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.2 

mg/ml BSA and 50% glycerol. 

Formulation 

1 μl of M.SssI protects 1 μg of genomic DNA 

from digestion with HpaII in 15 min at 37°C in 

50 μl of recommended reaction buffer. 

Storage 



402 

SgeI 

* SgeI cleaves DNA targets containing 5-methylcytosine on 

one or both DNA strands 

#ER2211 250 u 


 


Description 

The Thermo Scientific SgeI is a methylationdependent 

restriction enzyme which recognizes 

5-methylcytosine and cleaves modified site 

on one or both DNA strands at a N9/N13 

downstream the modified cytosine. 

Concentration 

3 u/μl 


Buffer SgeI at 37°C. 

Inactivation 

The SgeI is heat-inactivated at 65°C in 20 min. 


SgeI is supplied in: 

10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM 

NaCl, 1 mM DTT, 1 mM EDTA and 50% glycerol. 



Dcm: always cleaves DNA methylated by Dcm 

methyltransferase (p.175). 

CpG: cleaves targets overlapping with CpG 

methylated sequences (p.177). 


A minimum of 3 units of the enzyme is required 


lambda DNA in 16 hours. 

Applications 

Enrichment of undermethylated DNA. 

DNA Methylation analysis. 

Note 

 

methyltransferases will be a substrate 

for SgeI. 

 

may result in nonspecific cleavage. 

 

site are required for an efficient cleavage. 

 

complete digestion of methylated DNA 

depends on the number of SgeI recognition 

sites. Digestion products generated by 

target site cleavage may cause nonspecific 

cleavage by SgeI. Therefore, the amount 

of SgeI used in the reaction may require 

optimization. 

E.coli 

strain (#SD0041) can be used as a DNA 

cleavage efficiency control. SgeI cleaves 

all six dcm-methylated targets 

on pBR322 DNA.

CpG Methylated Human Genomic DNA 

#SD1131 15 μg 



Analysis Kit (MspI/HpaII) p.399 



EpiJET 5-hmC Analysis Kit p.400 

CpG Methylated Jurkat Genomic DNA 

#SD1121 15 μg 







Jurkat Genomic DNA 

#SD1111 15 μg 







Description 

Human genomic DNA enzymatically methylated 

with CpG Methyltransferase (M.SssI). Genomic 

DNA was isolated from blood samples from 

healthy male (HIV, HBV and HCV negative) 

individuals. Isolated genomic DNA was treated 

with CpG Methyltransferase, repurified and 

dissolved in TE buffer. 98% CpG methylation 

level confirmed by bisulfite sequencing. 

Description 

Human male genomic DNA from Jurkat 

cells enzymatically methylated with CpG 

Methyltransferase (M.SssI). Genomic DNA 

was isolated from Jurkat E6.1 cells (human 

acute T-cell leukemia, clone E6.1) grown to 

confluency in RPMI media supplemented with 

10% fetal bovine serum. Isolated genomic 

DNA was treated with CpG Methyltransferase, 

repurified and dissolved in TE buffer. 98% 

CpG methylation level confirmed by bisulfite 

sequencing. 

Description 

Human male genomic DNA purified from Jurkat 

cells. Genomic DNA was isolated from Jurkat 

E6.1 cells (human acute T-cell leukemia, clone 

E6.1) grown to confluency in RPMI media 

supplemented with 10% fetal bovine serum. 

Isolated genomic DNA is dissolved in TE buffer. 

Features 

Pure – DNA is free of inhibitors, proteins 

and RNA. 

Convenient – ready to use 100 ng/μl 


Compatible with downstream applications. 

Features 


and RNA. 




Complete methylation of cytosines in CpG 

dinucleotides. 

Applications 

As a control in the following: 

– Bisulfite sequencing, 

– Combined Bisulfite Restriction Analysis 

(COBRA), 

– Methylation-sensitive Single-Nucleotide 

Primer Extension (Ms-SNuPE). 

Features 


and RNA. 




Complete methylation of cytosines in CpG 

dinucleotides. 

Applications 




(COBRA), 



Applications 




(COBRA), 



Genomic DNA library construction. 

PCR, Southern blotting. 


404 



Nucleotides & Primers 

Deoxyribonucleoside Triphosphates (dNTPs), molecular biology grade .................406 

dNTP Sets and Mixes .....................................................................................................408 

dATP................................................................................................................................408 

dCTP ...............................................................................................................................408 

dGTP ...............................................................................................................................409 

dTTP ................................................................................................................................409 

dUTP ...............................................................................................................................409 

dITP ................................................................................................................................. 410 

Ribonucleoside Triphosphates (NTPs), molecular biology grade ...............................411 

NTP Set ............................................................................................................................411 

ATP ...................................................................................................................................411 

CTP .................................................................................................................................. 412 

GTP ................................................................................................................................. 412 

UTP.................................................................................................................................. 412 

Modified Nucleotides, molecular biology grade ............................................................ 413 

Fluorescein-12-dUTP ..................................................................................................... 413 

Biotin-11-dUTP ............................................................................................................... 414 

Aminoallyl-dUTP ............................................................................................................ 414 

Aminoallyl-UTP .............................................................................................................. 414 

dm6ATP ........................................................................................................................... 415 

dm4CTP ........................................................................................................................... 415 

dm5 Deoxyribonucleoside Triphosphates (dNTPs), molecular biology grade .................406 

dNTP Sets and Mixes .....................................................................................................408 

dATP................................................................................................................................408 

dCTP ...............................................................................................................................408 

dGTP ...............................................................................................................................409 

dTTP ................................................................................................................................409 

dUTP ...............................................................................................................................409 

dITP ................................................................................................................................. 410 

Ribonucleoside Triphosphates (NTPs), molecular biology grade ...............................411 

NTP Set ............................................................................................................................411 

ATP ...................................................................................................................................411 

CTP .................................................................................................................................. 412 

GTP ................................................................................................................................. 412 

UTP.................................................................................................................................. 412 

Modified Nucleotides, molecular biology grade ............................................................ 413 

Fluorescein-12-dUTP ..................................................................................................... 413 

Biotin-11-dUTP ............................................................................................................... 414 

Aminoallyl-dUTP ............................................................................................................ 414 

Aminoallyl-UTP .............................................................................................................. 414 

dm 

CTP ........................................................................................................................... 415 

Primers .............................................................................................................................. 416 

Primers for Sequencing ................................................................................................ 416 

M13/pUC Sequencing Primers ....................................................................................... 416 

pJET1.2 Sequencing Primers ......................................................................................... 416 

Transcription Promoter Primers ...................................................................................... 416 

Oligo(dT) 18 Primer .......................................................................................................... 417 

Random Hexamer Primer .............................................................................................. 417 

Exo-Resistant Random Primer ..................................................................................... 417 

6ATP ........................................................................................................................... 415 

dm4CTP ........................................................................................................................... 415 

dm5CTP ........................................................................................................................... 415 

Primers .............................................................................................................................. 416 

Primers for Sequencing ................................................................................................ 416 

M13/pUC Sequencing Primers ....................................................................................... 416 

pJET1.2 Sequencing Primers ......................................................................................... 416 

Transcription Promoter Primers ...................................................................................... 416 

Oligo(dT) 18 Primer .......................................................................................................... 417 

Random Hexamer Primer .............................................................................................. 417 

Exo-Resistant Random Primer ..................................................................................... 417 


406 

Deoxyribonucleoside Triphosphates (dNTPs), molecular biology grade 

Thermo Fisher Scientific is one of the few 

primary manufactures of nucleotides in the 

industry. Our dNTPs are supplied in aqueous 

solutions titrated to pH 7.0 with NaOH and 

are available individually, as a set of four 

separate nucleotides, or as nucleotide mixes 

at convenient concentrations for direct use in 

PCR, reverse transcription, labeling and other 

molecular biology applications. 

Thermo Scientific dNTP purity is greater than 99% confirmed by HPLC. 

Column – TOSOH TSK gel ODS – 100 V, 150 x 4.6 mm. 

Buffer A – 100 mM triethylamonium acetate, pH 7.0 

Buffer B – 60% acetonitrile/A 

Gradient – 0-25% B 

Flow rate – 1 ml/min 


Features 

Greater than 99% purity confirmed by HPLC. 

Free of trace contaminating nucleotides. 

Free of endo- and exodeoxyribonuclease, 

ribonuclease and nicking activities. 

Free of human and E.coli DNA. 

Highly stable – the neutral pH of the 

nucleotide solutions ensures stability during 

long-term storage: 

– stable for 3 years at -20°C, 

– stable after multiple freeze-thaw cycles, 

– 90-95% of dNTPs remain in triphosphate 

form after 7 weeks at room temperature, 

– 85-90% of dNTPs remain in triphosphate 

form after 30 cycles of PCR 

(1 min at 94°C; 3 min at 72°C). 

Application tested in: 

– long range PCR (40 kb), 

– cDNA synthesis and RT-PCR, 

– real-time PCR, 

– standard PCR, 

– high fidelity PCR. 

Application 

Thermo Scientific dNTPs can be used in 

most common molecular biology applications 

including PCR, real-time PCR, high fidelity and 

long PCR, LAMP-PCR, cDNA synthesis, RT-PCR, 

RDA, MDA, DNA labeling and DNA sequencing. 


Functionally tested in PCR with proofreading 

Pfu DNA Polymerase, exept dUTP, dITP and 

dNTP/dUTP Mix, which were tested with 

Taq DNA Polymerase. Greater than 99% purity 

of each component confirmed by HPLC. 

The absence of of endo-, exodeoxyribonuclease, 

ribonuclease and nicking activities confirmed by 

appropriate tests. Tested for absence of human 

and E.coli DNA. 

Storage 

Store at -20°C.

Fluorescence (norm) 

Cycle 

10 4 

10 3 

30 

20 

10 

0 4 

10 -5 

NTC 

8 12 16 20 

Cycle 

24 28 32 36 40 

1 10 10 

Amount, pg 

2 

10-4 10-3 10-2 10-1 103 104 Uniform and reproducible results in qPCR. 

Amplification of 10-fold serial dilutions of supercoiled plasmid DNA, 

(10 ng-0.1 fg), using the Maxima SYBR Green/ROX qPCR Master Mix 

(2X) (includes Thermo Scientific dNTPs) in duplicate reactions. Reactions 

were performed on the Eppendorf Mastercycler ep realplex instrument. 

The amplification plot and standard curve show the linearity across 9 

orders of magnitude. NTC is the non-template control. 

1μg 400ng 200ng 40ng 8ng 1.6ng 0.3ng 0.1ng 50pg 

M 1 2 3 4 5 6 7 8 9 M 

dNTP performance in two-step RT-PCR. 

Decreasing amounts of total mouse liver RNA were used for first-strand 

cDNA synthesis with RevertAid H Minus Reverse Transcriptase (Thermo 

Scientific), oligo(dT) 18 (#SO131) and dNTP Mix, 10 mM each (#R0191/2/3). 

After completion of the reaction, 2 μl of the mixture was used in 

subsequent PCR with Taq DNA Polymerase (Thermo Scientific) and PCR 

primers specific to the GAPDH gene. 

M – GeneRuler 100 bp Plus DNA Ladder (#SM0321). 

1-9 – RT-PCR products. 

M 1 2 3 4 5 M 

dNTP performance in amplification of long DNA 

fragments. 

Long DNA fragments were amplified in a 50 μl reaction 

mixture containing 2.5 u of the Thermo Scientific Long 

PCR Enzyme Mix, 1.25 ng of lambda DNA (#SD0011), 

0.2 mM of each dNTP (#R0241) and 1.5 mM of MgCl 2. 

M – DNA Ladder. 

1-5 – Long PCR products. 

1 kb RNA transcript 200 b RNA transcript 

M K1 A B C M K2 A B C M 

Evaluation of ribonuclease-free Thermo Scientific dNTPs. 

Three Thermo Scientific dNTP formulations (all at 1 mM concentration) 

were incubated with 1 μg of RNA transcript (200 b and 1 kb) for 4 hours 

at 37°C in 20 μl volume. The dNTP preparations are determined to be 

RNase free if there is no visible trace of RNA transcript degradation. 

M – RiboRuler Low Range RNA Ladder, ready-to-use (#SM1833). 

K1 – control RNA transcript 1 kb long. 

K2 – control RNA transcript 200 b long. 

A – with dNTP Mix, 2 mM each (#R0241). 

B – with dNTP Mix, 10 mM each (#R0191). 

C – with dNTP Set (#R0181). 


408 

dNTP Sets and Mixes 

Product Cat. # Concentration Amount 

dNTP Set 

dNTP Mix, 25 mM each 



dNTP/dUTP Mix* R0251 

dATP 

#R0141 25 μmol 

(0.25 ml of 100 mM solution) 

Formula 

C 10H 13N 5O 12P 3Na 3 


557.2 (acid form: 491.2) 

dCTP 

#R0151 25 μmol 


Formula 

C 9H 13N 3O 13P 3Na 3 


533.1 (acid form: 467.1) 


Description 

dATP (2’-deoxyadenosine 5’-triphosphate) is 

supplied as a 100 mM aqueous solution titrated 

to pH 7.0 with NaOH. 

Application 

For use in most common molecular biology 

applications including PCR, real-time PCR, 

high fidelity and long PCR, LAMP-PCR, cDNA 

synthesis, RT-PCR, RDA, MDA, DNA labeling 

and DNA sequencing. 

Description 

dCTP (2’-deoxycytidine 5’-triphosphate) is 



Application 



high fidelity and long PCR, LAMP-PCR, cDNA 

synthesis, RT-PCR, RDA, MDA, DNA labeling 

and DNA sequencing. 

General Characteristics 

max = 259 nm, 

= 15.4x10 3 M -1 x cm -1 (pH 7.0). 

Storage 



max= 271 nm, 

= 9.1x10 3 M -1 x cm -1 (pH 7.0). 

Storage 


Number of 

PCR applications 

(50 μl at 0.2 mM each) 

R0181 100 mM solutions of 

4x0.25 ml (4x25 μmol) 2500 

R0182 dATP, dTTP, dCTP and dGTP 4x1 ml (4x100 μmol) 10000 

R0186 

in separate tubes 

4x5 ml (4x500 μmol) 50000 

R1121 

1.0 ml (100 μmol total) 2500 

25 mM of each 

R1122 5x1.0 ml (500 μmol total) 12500 

R0191 

0.2 ml (8 μmol total) 200 

R0192 10 mM of each 

1.0 ml (40 μmol total) 1000 


R0241 

1.0 ml (8 μmol total) 200 

2 mM of each 


2 mM of each dATP, dCTP and dGTP 

4 mM of dUTP 

* not suitable for PCR with proofreading enzymes, enzyme mixes containing proofreading enzymes and for DreamTaq DNA Polymerase. 

1.0 ml (10 μmol total) 200

dGTP 

#R0161 25 μmol 


Formula 

C 10H 13N 5O 13P 3Na 3 


573.2 (acid form: 507.2) 

dTTP 

#R0171 25 μmol 


Formula 

C 10H 14N 2O 14P 3Na 3 


548.1 (acid form: 482.1) 

dUTP 

#R0133 25 μmol 


Formula 

C 9H 12N 2O 14P 3Na 3 


534.1 (acid form: 468.1) 

Description 

dGTP (2’-deoxyguanosine 5’-triphosphate) is 



Application 



high fidelity and long PCR, LAMP-PCR, 

cDNA synthesis, RT-PCR, RDA, MDA, DNA 

labeling and DNA sequencing. 

Description 

dTTP (2’-deoxythymidine 5’-triphosphate) is 



Application 



high fidelity and long PCR, LAMP-PCR, 

cDNA synthesis, RT-PCR, RDA, MDA, DNA 

labeling and DNA sequencing. 

Description 

dUTP (2’-deoxyuridine 5’-triphosphate) is 



Application 


applications including PCR, real-time 

PCR & RT-PCR with Taq DNA Polymerase, 

cDNA synthesis and primer extension. 


max= 253 nm, 

= 13.7x10 3 M -1 x cm -1 (pH 7.0). 

Storage 



max= 267 nm, 

= 9.6x10 3 M -1 x cm -1 (pH 7.0). 

Storage 



max= 262 nm, 

= 10.0x10 3 M -1 x cm -1 (pH 7.0). 

Storage 


Note 

 

proofreading enzymes or enzyme mixes 

containing proofreading enzymes. 


410 

dITP 

#R1191 25 μmol 


Formula 

C 10H 12N 4O 13P 3Na 3 


558.1 (acid form: 492.1) 


Description 

dITP (2’-deoxyinosine 5’-triphosphate) is 

supplied as a 100 mM aqueous solution 

titrated to pH 7.0 with NaOH. 

Application 


applications including PCR & RT-PCR with 

Taq DNA Polymerase, cDNA synthesis, 

SNP genotyping and primer extension. 


max= 249 nm, 

= 12.2x10 3 M -1 x cm -1 (pH 7.0). 

Storage 


Note 

 

proofreading enzymes or enzyme mixes 

containing proofreading enzymes.

Ribonucleoside Triphosphates (NTPs), molecular biology grade 

NTPs are manufactured at Thermo Fisher 

Scientific, one of a few primary manufacturers 

of nucleotides in the industry. They are provided 

individually or in a convenient set of ATP, CTP, 

GTP and UTP 100 mM solutions titrated to 

pH 7.0 with NaOH. 

Features 

Greater than 99% purity confirmed by HPLC. 

Free of trace contaminating nucleotides. 

Free of endo-, exodeoxyribonuclease, 

ribonuclease and nicking activities. 

Functionally tested in in vitro transcription. 

Highly stable – the neutral pH of nucleotide 

solutions ensures stability during long-term 

storage: 

– stable for 3 years at -20°C, 

– stable after multiple freeze-thaw cycles. 

NTP Set 

#R0481 4x25 μmol 

(4x0.25 ml of 100 mM solution) 

ATP 

#R0441 25 μmol 


Formula 

C 10H 13N 5O 13P 3Na 3 


573.1 (acid form: 507.2) 

Thermo Scientific vendor A vendor B 

M 1 2 3 4 5 6 7 8 9 M 

NTP performance in high yield in vitro trancription. 

RNA transcripts were generated using Thermo Scientific NTPs and 

NTPs from two other vendors in high yield in vitro transcription reactions 

containing 1 μg of DNA template. 

M – RiboRuler High Range RNA Ladder, ready-to-use (#SM1823). 




Description 

A convenient set of 100 mM solutions of each 

ATP, CTP, GTP and UTP. 

Applications 



siRNA synthesis 

aRNA synthesis 

Description 

ATP (adenosine 5’-triphosphate) is supplied as 

a 100 mM aqueous solution titrated to 


Applications 





Ligation 

Phosphorylation 

Storage 



max= 259 nm, 

= 15.4x10 3 M -1 x cm -1 (pH 7.0). 

Storage 



412 

CTP 

#R0451 25 μmol 


Formula 

C 9H 13N 3O 14P 3Na 3 


549.1 (acid form: 483.1) 

GTP 

#R0461 25 μmol 


Formula 

C 10H 13N 5O 14P 3Na 3 


589.2 (acid form: 523.2) 

UTP 

#R0471 25 μmol 


Formula 

C 9H 12N 2O 15P 3Na 3 


550.1 (acid form: 484.1) 


Description 

CTP (cytidine 5’-triphosphate) is supplied as 



Applications 





Description 

GTP (guanosine 5’-triphosphate) is supplied as 



Applications 





Description 

UTP (uridine 5’-triphosphate) is supplied as 



Applications 






max= 271 nm, 

= 9.0x10 3 M -1 x cm -1 (pH 7.0). 

Storage 



max= 253 nm, 

= 13.7x10 3 M -1 x cm -1 (pH 7.0). 

Storage 



max= 262 nm, 

= 10.0x10 3 M -1 x cm -1 (pH 7.0). 

Storage 

Store at -20°C.

Modified Nucleotides, molecular biology grade 

Fluorescein-12-dUTP 

#R0101 25 nmol 

(25 μl of 1 mM solution) 

Formula 

C 39H 41N 4O 21P 3 


994.7 

Description 

Fluorescein-12-dUTP (fluorescein-5(6)carboxaminocaproyl-[5-{3-aminoallyl}-2’deoxyuridine-5’-triphosphate]) 

is supplied as 

a 1 mM aqueous solution. 

Fluorescein-12-dUTP can be enzymatically 

incorporated into DNA with Reverse 

Transcriptases, Taq DNA Polymerase, 

phi29 DNA Polymerase, Klenow Fragment, exo – , 

Klenow Fragment and DNA Polymerase I. 

Fluorescence excitation 

excitation 

emission 

Normalized excitation-emission spectra of 

Fluorescein-12-dUTP. 

Fluorescence excitation 

400 450 500 550 600 650 700 

Wavelength, nm 

Application 

Enzymatic non-radioactive labeling of DNA 

during cDNA synthesis, PCR, nick-translation, 

random primed labeling or primer extension. 


Ex max= 495 nm; Em max= 520 nm (pH 9.0). 

max= 495 nm,= 70.0x10 3 M -1 x cm -1 (pH 9.0). 

Storage 

Store at -20°C in the dark. 

Ex max – excitation maximum; Em max – emission maximum. 


» 6.2.2. Non-radioactive random-primed DNA 

labeling p.350 



» 6.5.2. Synthesis of non-radioactively 

labeled cDNA p.351 


414 

Biotin-11-dUTP 

#R0081 50 nmol 

(50 μl of 1 mM solution) 

Formula 

C 28H 41N 6O 17P 3SNa 3 


927.6 (acid form: 861.6) 

Aminoallyl-dUTP 

#R0091 1 μmol 


#R1101 2.5 μmol 


Formula 

C 12H 17N 3O 14P 3Na 3 


589.2 (acid form: 523.2) 

Aminoallyl-UTP 

#R1091 2.5 μmol 


Formula 

C 12H 17N 3O 15P 3Na 3 


605.2 (acid form: 539.2) 


Description 

Biotin-11-dUTP (biotin--aminocaproyl-[5- 

{3-aminoallyl}-2’-deoxyuridine-5’-triphosphate]) 

is supplied as a 1 mM aqueous solution titrated 


Biotin-11-dUTP can be enzymatically 


Transcriptases, Taq DNA Polymerase, 

phi29 DNA Polymerase, Klenow Fragment, exo – , 

Klenow Fragment and DNA Polymerase I. 

Description 

Aminoallyl-dUTP (5-[3-aminoallyl]-2’deoxyuridine-5’-triphosphate) 

is supplied as 

10 mM and 50 mM aqueous solutions titrated 


Aminoallyl-dUTP can be enzymatically 


Transcriptases, Taq DNA Polymerase, phi29 

DNA Polymerase, Klenow Fragment, Klenow 

Fragment, exo – and DNA Polymerase I. The 

resulting amine-containing DNA can be 

subsequently labeled with any amine-reactive 

fluorescent dye, biotin or hapten. 

Description 

Aminoallyl-UTP (5-[3-aminoallyl]-2’-uridine-5’triphosphate) 

is supplied as a 50 mM aqueous 

solution titrated to pH 7.0 with NaOH. 

Aminoallyl-UTP can be enzymatically 

incorporated into RNA with T7, T3 and SP6 RNA 

polymerases. The resulting amine-containing 

RNA can be subsequently labeled with any 

amine-reactive fluorescent dye, biotin or 

hapten. 

Application 

Enzymatic non-radioactive labeling of DNA 

during PCR, nick-translation, cDNA synthesis, 

random primed labeling or primer extension. 


max= 240 nm, =10.7x103 M-1 x cm-1 (pH 7.0) 

max= 289 nm,= 7.1x103 M-1 x cm-1 (pH 7.0). 

Storage 


Application 

Enzymatic indirect non-radioactive labeling 

of DNA during cDNA synthesis, PCR, nicktranslation, 

random primed labeling or primer 

extension. 


max = 240 nm, =11.9x103 M-1 x cm-1 (pH 7.0) 

max = 290 nm, = 7.8x103 M-1 x cm-1 (pH 7.0). 

Storage 


Application 

Enzymatic indirect non-radioactive labeling of 

RNA during in vitro transcription. 


max = 240 nm, =11.9x10 3 M -1 x cm -1 (pH 7.0) 

max = 290 nm, = 7.8x10 3 M -1 x cm -1 (pH 7.0). 

Storage 

Store at -20°C.

dm 6 ATP 

#R0501 1 μmol 


Formula 

C 11H 15N 5O 12P 3Na 3 


573.2 (acid form: 507.2) 

dm 4 CTP 

#R0421 1 μmol 


Formula 

C 10H 15N 3O 13P 3Na 3 


547.1 (acid form: 481.1) 

dm 5 CTP 

#R0431 1μmol 

(0.1ml of 10 mM solution) 

Formula 

C 10H 15N 3O 13P 3Na 3 


547.1 (acid form: 481.1) 

Description 

dm 6 ATP (2’-deoxy-N6-methyladenosine 

5’-triphosphate) is supplied as a 10 mM 

aqueous solution titrated to pH 7.0 with NaOH. 

dm 6 ATP can be enzymatically incorporated 

into DNA with Taq DNA Polymerase or Klenow 

Fragment. 

Description 

dm 4 CTP (2’-deoxy-N4-methylcytidine 


aqueous titrated to pH 7.0 with NaOH. 

dm 4 CTP can be enzymatically incorporated 


Fragment. 

Description 

dm 5 CTP (2’-deoxy-5-methylcytidine 


aqueous solution titrated to pH 7.0 with NaOH. 

dm 5 CTP can be enzymatically incorporated 


Fragment. 

Application 

Synthesis of methylated DNA. 


max = 265 nm, 

= 15.4x103 M-1 x cm-1 (pH 7.0). 

Storage 


Application 



max = 274 nm, 

= 13.6x103 M-1 x cm-1 (pH 7.0). 

Storage 


Application 



max = 279 nm, 

= 8.77x103 M-1 x cm-1 (pH 7.0). 

Storage 



416 

Primers 

Primers for Sequencing 

M13/pUC Sequencing Primers Cat. # 

A260 units 

Amount 

μg μl pmol 

Concentration 

μg/ml μM 

M13/pUC sequencing primer (-20), 17-mer 

5’-d(GTAAAACGACGGCCAGT)-3’ 

SO100 0.1 3.3 60 600 56.6 10 

M13/pUC reverse sequencing primer (-26), 17-mer 

5’-d(CAGGAAACAGCTATGAC)-3’ 

SO101 0.1 3.3 60 600 56.6 10 


5’-d(GTTTTCCCAGTCACGAC)-3’ 

SO113 0.1 3.3 60 600 56.6 10 


5’-d(GCCAGGGTTTTCCCAGTCACGA)-3’ 

SO114 0.1 3.3 45 450 73.2 10 

M13/pUC reverse sequencing primer (-46), 24-mer 

5’-d(GAGCGGATAACAATTTCACACAGG)-3’ 

SO115 0.1 3.3 42 420 79.9 10 

pJET1.2 Sequencing primers Cat. # A260 units g l pmol g/ml m 

pJET1.2 forward sequencing primer, 23-mer 

5’-d(CGACTCACTATAGGGAGAGCGGC)-3’ 

SO501 0.2 6.7 87 870 76.6 10 

pJET1.2 reverse sequencing primer, 24-mer 

5’-d(AAGAACATCGATTTTCCATGGCAG)-3’ 

SO511 0.2 6.7 84 840 79.9 10 

Description 

Sequencing primers are single-stranded 

oligonucleotides with 5’-hydroxyl and 

3’-hydroxyl ends. The M13/pUC sequencing 

primers anneal to the region in the 5’-terminus 

of the lacZ gene. All primers are supplied as 

10 μM aqueous solutions. 

Transcription Promoter Primers Cat. # 

SP6 promoter sequencing primer, 18-mer 

5’-d(ATTTAGGTGACACTATAG)-3’ 

SP6 promoter sequencing primer, 24-mer 

5’-d(CATACGATTTAGGTGACACTATAG)-3’ 

T7 promoter sequencing primer, 20-mer 

5’-d(TAATACGACTCACTATAGGG)-3’ 


5’-d(ATTAACCCTCACTAAAG)-3’ 


5’-d(GCGCGAAATTAACCCTCACTAAAG)-3’ 

Description 

Sequencing primers are single-stranded 

oligonucleotides with 5’- and 3’-hydroxyl ends. 

The primers are complementary to the SP6, 

T3 or T7 RNA polymerase promoter regions 

respectively. Primers are supplied as 10 μM 

aqueous solutions. 


Applications 

M13/pUC Sequencing primers 

– Sequencing of DNA fragments inserted 

into the MCS within the lacZ gene of 

various cloning vectors, such as pUC19, 

pTZ19R, pTZ57R, M13mp18, 

pBluescript II (see Appendix for vector 

description). 

– Colony screening by PCR. 

pJET1.2 Sequencing primers 

– Sequencing of DNA fragments inserted 

into Eco32I site within the eco47IR gene 

of pJET1.2. 

– Colony screening by PCR. 

Amount Concentration 

A 260 units μg μl pmol μg/ml μM 

SO116 0.1 3.3 56 560 59.9 10 

SO117 0.1 3.3 42 420 79.9 10 

SO118 0.1 3.3 50 500 66.6 10 

SO119 0.1 3.3 60 600 56.6 10 

SO120 0.1 3.3 42 420 79.9 10 

Application 

Sequencing of DNA fragments located 

downstream from the corresponding RNA 

polymerase promoter sequences in common 

cloning vectors, such as pTZ19R, pTZ57R, 

pBluescript II (see Appendix for vector 

description). 

Storage 


Storage 


Note 

 

that contain truncated, but still fully 

functional promoters.

Oligo(dT) 18 Primer 

#SO131 60 μl (30 μg) 

#SO132 120 μl (60 μg) 

Random Hexamer Primer 

Description 

The Oligo(dT) 18 Primer is a synthetic singlestranded 

18-mer oligonucleotide with 5’- and 

3’-hydroxyl ends. 

The primer is supplied as a ready-to-use, 

20X concentrated aqueous solution. 

#SO142 120 μl (24 μg) Description 

The Random Hexamer Primer is a mixture of 

single-stranded random hexanucleotides with 

5’- and 3’-hydroxyl ends. 

The primer is supplied as a ready-to-use, 


Exo-Resistant Random Primer 

5’-NpNpNpNpNp S Np S N-3’ 

N=G, A, T or C 

#SO181 100 μl 

References 

1. Skerra, A., Phosphorothioate primers improve the 

amplification of DNA sequences by DNA polymerases 

with proofreading activity, Nucleic Acids Res., 20, 

3551-3554, 1992. 

2. Dean, F.B., et al., Comprehensive human genome 

amplification using multiple displacement amplification, 

Proc. Natl. Acad. Sci., 99, 5261-5266, 2002. 

3. Dean, F.B., et al., Rapid amplification of plasmid and 

phage DNA using phi29 DNA polymerase and multiplyprimed 

rolling circle amplification, Genome Res., 11, 

1095-1099, 2001. 






activity, Addendum, Anal. Biochem., 137, 266-267, 1984. 

6. Mackey, J., et al., Use of random primer extension for 

concurrent amplification and nonradioactive labeling of 

nucleic acids, Anal. Biochem., 212, 428-435, 1993. 

Description 

The Exonuclease-Resistant Random Primer 

is a mixture of single-stranded random 

oligonucleotides used for highly efficient random 

priming of various DNA synthesis reactions. This 

primer has two 3’-terminal phosphorothioate 

(PTO) modifications that are resistant to the 

3’5’ exonuclease activity of proofreading 

DNA polymerases (1), such as Klenow Fragment 

and phi29 DNA Polymerase. It also has 

5’- and 3’-hydroxyl ends. The Exo-Resistant 

Random Primer is supplied as a ready-to-use, 


Application 

First strand cDNA synthesis. 

Concentration 

100 μM (0.5 μg/μl) 

Storage 


Application 

First strand cDNA synthesis. 

Concentration 

100 μM (0.2 μg/μl) 

Storage 


Features 

3’-terminal PTO modifications protect the 

primer from 3’5’ exonuclease degradation 

and ensure efficient priming of DNA synthesis. 

Applications 

Strand displacement amplification of genomic 

DNA (2), plasmids and phage DNA (3). 

DNA labeling by random priming (4-6). 

Concentration 

500 μM (1.1 μg/μl) 

Storage 



418 


Thermo Scientific Reagents 

Water .................................................................................................................................. 420 

Water, nuclease-free ..................................................................................................... 420 

DEPC-treated Water ...................................................................................................... 420 

Reducing Agent ............................................................................................................... 420 

DTT.................................................................................................................................. 420 

Aqueous Salt Solutions .................................................................................................. 421 

0.5 M EDTA, pH 8.0......................................................................................................... 421 

25 mM MgCl 2 .................................................................................................................. 421 

3 M Sodium Acetate Solution, pH 5.2, molecular biology grade ..................................... 421 

BSA Solution..................................................................................................................... 422 

Bovine Serum Albumin (BSA), molecular biology grade ................................................. 422 

Carriers for Precipitation of Nucleic Acids ................................................................ 423 

Glycogen......................................................................................................................... 423 

Glycogen, molecular biology grade ................................................................................. 423 

Glycogen, RNA grade .................................................................................................... 423 

Enzyme Substrates and Associated Reagents ......................................................... 424 

IPTG ................................................................................................................................ 424 

X-Gal ...............................................................................................................................424 

X-Gluc ............................................................................................................................. 425 

5-Fluoroorotic Acid ....................................................................................................... 425 

BCIP-T ............................................................................................................................. 426 

NBT ................................................................................................................................. 426 

Agarose ............................................................................................................................. 427 

TopVision Agarose ......................................................................................................... 427 

TopVision Agarose ......................................................................................................... 427 

TopVision Low Melting Point Agarose ............................................................................. 427 


420 

Water 


#R0581 4x1.25 ml 

#R0582 30 ml 


#R0603 5x1 ml 

#R0601 30 ml 

Reducing Agent 

DTT 

#R0861 5 g 

#R0862 25 g 

Formula 

C 4H 10O 2S 2 


154.25 


Description 

Deionized and 0.22 μm membrane-filtered 

nuclease-free water. Ideal for all molecular 

biology applications. 

Description 

 

and 0.22 μm membrane-filtered water. Ideal for 

applications involving RNA. 

Feature 

Free of endo-, exodeoxyribonucleases and 

ribonucleases. 

Description 

DTT (DL-Dithiothreitol; Cleland’s reagent) is 

used to stabilize enzymes and other proteins 

which possess free sulfhydryl groups and has 

been shown to restore activity lost by oxidation 

of these groups in vitro. DTT quantitatively 

reduces disulfide bonds and maintains 

monothiols in a reduced state (1). 

At a 0.1 M final concentration DTT is also widely 

used for disruption of protein disulfide bonds in 

SDS-polyacrylamide gel electrophoresis. 

Feature 

Free of endo-, exodeoxyribonucleases, 

ribonucleases and proteases. 

Features 

Free of endo-, exodeoxyribonucleases and 

ribonucleases. 

Human and E.coli DNA free. 

Storage 

Store at -20°C or at room temperature. 

Storage 


Note 

 

PCR or in vivo experiments (1). 

Reference 

 


Laboratory Press, Cold Spring Harbor, New York, 

7.84, 2001. 

Applications 

 

 

Protein electrophoresis. 

Storage 

Store at 4°C or at room temperature. 

Note 

 

not stable in solution. To prepare a 1 M DTT 

solution, dissolve 1.55 g of DTT powder in 

 

nuclease-free, #R0581). 

Reference 

 

SH groups, Biochemistry, 3, 480-2, 1964.

Aqueous Salt Solutions 

0.5 M EDTA, pH 8.0 

#R1021 5x1 ml 

25 mM MgCl 2 

Description 

An aqueous solution of 0.22 μm membrane- 

 

sodium salt. Ideal for biochemistry or molecular 

biology applications that require a chelator for 

 

enzymatic reactions by forming complexes with 

 

 

 

degradation of nucleic acids by metal 


Usage recommendations 

 

 

– mix well. 

 

PCR Purification Kit (#K0701) or phenol/chloroform extraction with subsequent alcohol precipitation. 

Note 

 

thermally inactivated, if possible. 

Applications 

Termination of metal dependent enzymatic 

reactions. 

Protection of DNA against metal dependent 

nucleases. 

Storage 

Store at 4°C. 

#R0971 4x1.25 ml Description 

Applications 

An aqueous solution of 0.22 μm 

PCR. 

membrane-filtered magnesium chloride, used 

for optimization of magnesium ion concentration 

in PCR reactions. 

Feature 

Free of endo-, exodeoxyribonucleases 

and ribonuclease. 

Storage 


3 M Sodium Acetate Solution, pH 5.2, molecular biology grade 

#R1181 5x1 ml Description 

Storage 

3 M Sodium Acetate Solution, pH 5.2 is 0.22 μm 

membrane filtered, sterile and nuclease free. 

Applications 

DNA precipitation. 

RNA precipitation. 

Store at 4°C. 


422 

BSA Solution 

Bovine Serum Albumin (BSA), molecular biology grade 

#B14 5 mg (20 mg/ml) Description 

Concentration 

Bovine Serum Albumin (BSA) is used for 20 mg/ml 

stabilization of enzymes during storage and 

for enzymatic reactions where the absence 

of nucleases is essential. BSA increases PCR 

yields from low purity templates. It also prevents 

adhesion of enzymes to reaction tubes and tip 

surfaces. 

Features 

Non-modified (non-acetylated). 

V-fraction. 

Applications 

 

PCR with low purity templates, e.g., mouse 

tail DNA. 

Restriction enzyme digestion. 


Approximately 66 kDa. 


 

10 mM Tris-HCl (pH 7.4 at 25°C), 100 mM KCl, 

 

Storage 



Carriers for Nucleic Acid Precipitation 

Glycogen 

Glycogen, molecular biology grade 

#R0561 2x0.25 ml 

(20 mg/ml aqueous solution) 

Glycogen, RNA grade 

#R0551 2x0.1 ml 

(20 mg/ml aqueous solution) 

Precipitation yield, % 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

Glycogen-free Glycogen 

1 μg/μl 

17 nt DNA oligonucleotide 


0.05 μg/μl 

200 bp DNA fragment 

References 

1. Tracy, S., Improved rapid methodology for the isolation 

of nucleic acids from agarose gels, Prep. Biochem., 11, 

251-268, 1981. 

2. Helms, C., A new method for puryfying lambda DNA from 

phage lysates, DNA, 4, 39-49, 1985. 

3. Hengen, P. N., Methods and reagents – Carriers for 

precipitating nucleic acids, TIBS, 21, 224-225, 1996. 

4. 


Laboratory Press, Cold Spring Harbor, New York, A8. 

12-8.13, 2001. 

Description 

Glycogen is a highly purified polysaccharide 

derived from oysters that acts as an inert 

carrier to significantly increase the recovery of 

nucleic acids by alcohol precipitation. Glycogen 

is insoluble in ethanol and forms a precipitate 

that traps nucleic acids. During centrifugation a 

visible pellet is formed which greatly facilitates 

handling of the precipitated nucleic acids. 

Glycogen quantitatively precipitates nucleic 

acids from diluted solutions with a higher 

efficiency than tRNA, linear polyacrylamide or 

sonicated DNA (1-4). 

 

solutions that allow for fast and efficient DNA or 

RNA recovery. 

Glycogen, molecular biology grade (#R0561), 

recommended for DNA precipitation. 

Glycogen, RNA grade (#R0551), can be used 

for both RNA and DNA precipitation. 


0.05 μg/μl 

100 nt RNA transcript 

Effect of glycogen on precipitation of nucleic acids. 

A 17 nt DNA oligonucleotide was precipitated from 2 pg/μl solution without glycogen and with 1 μg/μl final 

concentration of Glycogen, molecular biology grade (#R0561). A 200 bp DNA fragment was precipitated from 

50 pg/μl solution without glycogen and with 0.05 μg/μl final concentration of Glycogen, molecular biology grade 

(#R0561). 100 nt RNA transcript was precipitated from 50 pg/μl solution, without Glycogen and with 

0.05 μg/μl final concentration of Glycogen, RNA grade (#R0551). All precipitations were performed with 2.5X 

volumes of ethanol and 0.3 M sodium acetate at room temperature for 5 minutes. The concentration of the 

nucleic acids was measured spectrophotometrically. 

Features 

Ideal for highly efficient recovery of 

oligonucleotides (>8 bases) and low amounts 

of DNA/RNA (>20 pg) from diluted solutions. 

Forms clearly visible pellets. 

Does not interfere with enzymatic reactions*. 

Does not interfere with gel electrophoresis of 

nucleic acids. 

Does not interfere with spectrophotometrical 

determination of DNA/RNA concentration, 

(A 260/A 280 measurements). 

Compatible with DNA transformation and 

in vitro transfection. 

Source 

Oysters. 


Glycogen molecules are highly branched 

structures which are composed of thousands of 

glucose molecules bonded to each other. 

The molecular weight of the largest individual 

glycogen molecule appears to be 8 million 

containing about 50000 glucose molecules. 

Storage 


* At a final concentration up to 8 μg/μl, Glycogen does not 

interfere with PCR, DNA sequencing, DNA digestion by 

endonucleases, ligation, cDNA synthesis, DNA labeling, 

in vitro transcription or bacterial transformation. At a final 

concentration up to 0.4 μg/μl, Glycogen does not affect in 

vitro in vitro 

Transfection Reagent. 


» 4.5. Nucleic acid precipitation from diluted 

solutions with glycogen p.323 


424 

Enzyme Substrates and Associated Reagents 

IPTG 

IPTG Solution, ready-to-use 

#R1171 10x1.5 ml 

(100mM aqueous solution) 

IPTG, dioxane-free 

#R0391 1 g 

#R0392 5 g 

#R0393 25 g 

Formula 

C 9H 18O 5S 


238.3 

X-Gal 

X-Gal Solution, ready-to-use 

#R0941 10 ml (20 mg/ml) 

X-Gal 

#R0401 0.5 g 

#R0402 2x1 g 

#R0404 1 g 

Formula 

C 14H 15BrClNO 6 


408.6 


» 3.7.2. Preparation of X-Gal/IPTG LB agar 

plates for blue/white colony screening p.296 


Description 

IPTG (isopropyl--D-thiogalactopyranoside) 

is a highly stable synthetic analog of lactose 

that inactivates the lac repressor and induces 

synthesis of -galactosidase, an enzyme that 

promotes lactose utilization. IPTG is used to 

induce expression of cloned genes under 

control of the lac operon (1). It is used in 

conjunction with X-Gal (#R0941) to determine 

the lac phenotype in blue/white colony assay. 

Ready-to-use IPTG is a 100mM non-toxic, 

stable, 0.22 μm membrane-filtered aqueous 

solution specially formulated for direct use in 

blue/white colony screening. 

Applications 

Blue/white colony screening to distinguish 

recombinant (white) from non-recombinant 


Induction of gene expression under control of 

the lac promoter. 

Description 

X-Gal (5-bromo-4-chloro-3-indolyl--Dgalacto-pyranoside) 

is an inert chromogenic 

substrate for -galactosidase which hydrolyzes 

X-Gal into colorless galactose and 4-chloro- 

3-brom-indigo, forming an intense blue 

precipitate. Induction of the lacZ gene with 

IPTG leads to the hydrolysis of X-Gal and to the 

development of blue colonies 

(see the scheme below). 

Ready-to-use X-Gal Solution is a stable 

0.22 μm membrane filtered aqueous 

solution specially formulated for direct use in 

conjunction with IPTG (#R1171) for blue/white 

colony screening. 

Applications 

Blue/white colony screening to distinguish 

recombinant (white) from non-recombinant 


Visualization of -galactosidase reporter 

gene expression in transfected 

eukaryotic cells. 

X-Gal 

(colorless) 

-gal 

galactose 

(colorless) + 

4-Cl-3-Br-indigo 

(deep blue) 

Storage 

IPTG Solution, ready-to-use (#R1171) 

store at -20°C. 

IPTG, dioxane-free (#R0391/2/3) store at 4°C. 

Note 

 

stock solution in water is recommended. 

 

final IPTG concentration in LB (Luria Broth) 

media. 

Reference 

 



1.124-1.125, A1.27, 2001. 

Detection of-galactosidase activity 

in immunological and histochemical 

procedures. 

Storage 

X-Gal Solution, ready-to-use (#R0941) is 

stable for at least 12 months at 4°C or at room 

temperature in the original vial. 

X-Gal (#R0401, #R0402, #R0404) should be 

stored at -20°C in the dark. 

Note 

 

stock solution in dimethylformamide (DMF) 

 

DMF dissolves some plastic materials. Avoid 

adding DMF containing solutions to plastic 

Petri dishes. 

 

containing DMF to completely dry before 

plating bacteria on agar plates. 

Reference 

 

Manual, the Third edition, Cold Spring Harbor Laboratory Press, 

Cold Spring Harbor, New York, 1.124-1.125, A1.27, 2001.

X-Gluc 

#R0851 0.1 g 

#R0852 0.5 g 

Formula 

C 14H 13BrClNO 7 x C 6H 13N 


521.8 

References 

1. 



16.42, 2001. 

2. 

the GUS gene fusion system, plant Mol Biol Rep, 

5, 387-405, 1987. 

5-Fluoroorotic Acid 

#R0811 1 g 

#R0812 5 g 

Formula 

C 5H 3FN 2O 4 x H 2O 


192.1 

orotine-5’-monophosphate 

dicarboxylase (OMP) 

5’-Fluoroorotic 

Acid 

fluorouracil 

(toxic) 

References 

1. Bartel, P.L. and Fields, S., Yeast 2-Hybrid System, Oxford 

University Press, New York, 7, 109-147, 1997. 

2. Kaiser, C., et al., Methods in Yeast Genetics, Cold spring 

Harbor Laboratory Press, Cold Spring Harbor, NY, 1994. 

Description 

X-Gluc (5-bromo-4-chloro-3-indolyl--D-glucu 

ronic acid, cyclohexylammonium salt) is a 

substrate for -glucuronidase (GUS) which 

is encoded by gusA, a widely used reporter 

gene. Glucuronidase cleaves X-Gluc to produce 

colorless glucuronic acid and an intense blue 

precipitate of chloro-bromoindigo (see the 

scheme below). 

X-Gluc 

(colorless) 

GUS 

glucuronic acid 

(colorless) 

4-Cl-3-Br-indigo 

(deep blue) 

Histochemical Detection of GUS (-glucoronidase) (2) 

Staining buffer 

+ 

Applications 

Detection of GUS expression in plant cells 

and tissues (1) (see protocol below). 

Detection of infections caused by E.coli. 

Detection of bacterial contamination in food 

and water samples. 

Storage 

Store in the dark at -20°C. 

Note 

 

stock solution in dimethylformamide or 

dimethylsulfoxide. 

Stock solutions Volume per 1 ml staining buffer Final concentration 

830 μl 

1 M sodium phosphate (pH 7.0) 100 μl 0.1 M 

20 μl 10 mM 

10 μl 

50 mM K3Fe(CN)6 20 μl 1 mM 

0.1 M X-Gluc (50 mg/ml) in dimethylformamide 20 μl 2 mM 

Staining procedure for cells and tissues: 

Remove media from the cells or tissue and add fresh staining buffer. 

1. Incubate for 12-24 hours at 37°C, remove the staining buffer. 

2. 

3. Count dark blue cells. 

Description 

5-Fluoroorotic Acid (5-fluorouracil-6carboxylic 

acid monohydrate; 5-FOA) is 

used in yeast molecular genetics to detect 

expression of the URA3 gene, which encodes 

orotine-5’-monophosphate (OMP) dicarboxylase. 

Yeast with an active URA3 gene (Ura+) converts 

5-FOA to fluorodeoxyuracil, which is toxic to 

cells (see the scheme below). Yeast strains 

carrying a mutation in the URA3 gene grow 

in the presence of 5-FOA if the media is 

supplemented with uracil. 5-FOA is frequently 

used in experiments with Saccharomyces 

cerevisiae (URA3), Schizosaccharomyces pombe 

(URA4 and URA5), Candida albicans (URA3) and 

E.coli (pyrF). 

Applications 

Detection of URA3 gene expression in yeast. 

Construction of yeast 2-hybrid libraries (1). 

Other areas of yeast genetics including 

gene replacement, analysis of transposable 

genetic elements and identification of gene 

mutations (2). 

Storage 


Note 

 

 

150 mg/ml. 

 

solution in dimethylsulfoxide. 

Usage recommendations 

For selection of Ura-minus cells, add 5-FOA powder or sterile 100 mg/ml 5-FOA stock solution to the yeast 

growth media at final concentration of 1 mg/ml. The reagent can be added to the media prior to or after 

sterilization. Ura-plus cells will not grow in media supplemented with 1 mg/ml of 5-FOA and 20 μg/ml uracil. 


426 

BCIP-T 

#R0821 1 g 

#R0822 5 g 

Formula 

C 8H 6NO 4BrClP x C 7H 9N 


433.6 

Reference 

 

Manual, the Third edition, Cold Spring Harbor Laboratory 

Press, Cold Spring Harbor, New York, 16.42, 2001. 

NBT 

#R0841 1 g 

#R0842 5 g 

Formula 

C 40H 30N 10O 6Cl 2 


817.7 




Description 

BCIP-T (5-bromo-4-chloro-3-indolyl phosphate, 

p-toluidine salt) is a chromogenic substrate for 

alkaline phosphatase used in conjunction with 

the oxidant NBT (nitro blue tetrazolium, #R0841) 

to enhance blue color development. 

Alkaline phosphatase catalyses the removal 

of a phosphate group from BCIP-T, generating 

a product that oxidizes and dimerizes 

to dibromodichloro indigo. The reducing 

equivalents produced during the dimerization 

reaction reduce NBT to an insoluble purple dye, 

diformazan (1). 

BCIP-T + NBT 

(colorless) 

alkaline 

phosphatase 

dibromodichloro 

indigo 

Description 

NBT (nitro blue tetrazolium) is an oxidant used in 

conjunction with BCIP or BCIP derivatives (BCIP 

T, #R0821), to enhance blue color development 

during detection of alkaline phosphatase 

activity. It is suitable for immunohistochemistry 

and cytochemistry. NBT is also a substrate for 

oxidases and dehydrogenases. 

+ 

Applications 

 

– Southern blotting, 

– 

– Northern blotting, 

– dot/slot blots, 

– plaque and colony screening. 

Storage 


Note 

 

solution in dimethylformamide. 

diformazan 

(deep purple) 

Applications 

 

– Southern blotting, 

– 

– Northern blotting, 

– dot/slot blots, 

– plaque and colony screening. 

Storage 

Store at 4°C or at -20°C in the dark. 

Note 

 

 

Preparation of BCIP-T/NBT substrate solution 

Add the following components to 10 ml of the Alkaline Phosphatase (AP) buffer (100 mM Tris-HCl (pH 9.5), 

100 mM NaCl and 10 mM MgCl 2 

BCIP-T (50 mg/ml in dimethylformamide) 33 μl 

NBT 44 μl 

Note 

 

Prepare fresh developing solution and use within an hour. 

 

2 years when stored in the dark at -20°C.

Agarose 



#R0491 100 g 

#R0492 500 g 

TopVision Low Melting Point 

Agarose 

#R0801 25 g 


Agarase p.319 

 

 

 





A B 

1 2 3 4 5 6 

Description 

TopVision Agarose is highly purified and suitable 

for analytical and preparative electrophoresis 

of nucleic acids. TopVision Low Melting Point 

Agarose is used for in-gel enzymatic processing 

experiments and for nucleic acid recovery from 

 

Features 

Can be used at concentrations between 

Agarose) and 

Low Melting Point 

Agarose) in all typical buffer systems. 

DNA recovered from preparative gels can be 

used for different applications (restriction, 

ligation, etc.). 

Low DNA/RNA binding. 

 

DNase, RNase free. 

Suitable for RNA analysis. 

A. Separation of Thermo Scientific DNA Ladders in TopVision Agarose and TopVision Low Melting Point Agarose. 

Electrophoresis conditions: 1% gel, 0.5 μg/lane, 1X TAE, 7 V/cm, 40 min. 

1 – GeneRuler DNA Ladder Mix, ready-to-use (#SM0333). 

2 – GeneRuler 1kb DNA Ladder, ready-to-use (#SM0313). 

3 – GeneRuler 1kb Plus DNA Ladder, ready-to-use (#SM1333). 

4 – ZipRuler 

5 – ZipRuler 

6 – GeneRuler 

B. Separation of Thermo Scientific RiboRuler RNA Ladders in TopVision Agarose and 

TopVision Low Melting Point Agarose. 

 

1 – RiboRuler 

2 – RiboRuler 

bases 

1000 

800 

600 

400 

300 

200 

100 

1 2 

bases 

6000 

4000 

3000 

2000 

1500 

1000 

500 

200 

A 

1 2 3 4 5 6 

Applications 

Analytical electrophoresis of nucleic acids. 

Preparative electrophoresis. 

Blotting assays. 

Properties for TopVision Agarose 

 

2 2 

 

 

Properties for TopVision Low 

Melting Point Agarose 

 

>250g/cm2 

 

 

TopVision Low Melting Point Agarose 

B 

bases 

1000 

800 

600 

400 

300 

200 

100 

1 2 

bases 

6000 

4000 

3000 

2000 

1500 

1000 

500 

200 


» 7.2. Preparation of agarose gels for 







428 

Appendix 

Bacterial Strain Genotypes ................................................................................. 429 

Genetic Markers ................................................................................................. 430 

E.coli Host Restriction and Modification Systems ............................................ 431 

Phage and Plasmid DNA Maps ........................................................................... 432 

pJET1.2 ...........................................................................................................................432 

Phage Lambda ...............................................................................................................434 

X174 .............................................................................................................................435 

M13mp18, M13mp19 ......................................................................................................437 

pBR322 ...........................................................................................................................439 

pUC18, pUC19 ................................................................................................................. 441 

pUC57 .............................................................................................................................443 

pTZ19R, pTZ19U .............................................................................................................445 

pTZ57R ............................................................................................................................ 447 

pBluescript II KS(+/-), pBluescript II SK(+/-) .............................................................449 

pACYC177 .......................................................................................................................453 

pACYC184 .......................................................................................................................455 

Number of Recognition Sites in DNA Molecules ..................................................................457 

Technical Data .................................................................................................... 461 

Genome Sizes ...................................................................................................................461 

DNA Migration in Agarose and Polyacrylamide Gels ............................................................462 

DNA Base Pairs ................................................................................................................462 

Codons and Assigned Amino Acids ....................................................................................462 

Physical Properties of Some Common Radioisotopes ..........................................................462 

Amino Acids .....................................................................................................................463 

Concentrations of Acids and Bases ....................................................................................464 

Physical Constants of the Nucleoside Triphosphates and Related Compounds .....................464 

SI Unit Prefixes .................................................................................................................464 

Common Conversions of Nucleic Acids ..............................................................................465 

Common Conversions of Oligonucleotides ..........................................................................465 

Melting Temperature of Duplex DNA and Oligonucleotides ..................................................465 

Temperature Dependence of the pH of 50 mM Tris-HCl Solutions .......................................465 

General Physical Constants ...............................................................................................465 

Temperature Dependence of the pH for Commonly Used Buffers ........................................466 

Decay Factors for Calculating the Amount of Radioactivity ..................................................466 

Commonly Used Media, Stock Solutions and Buffers ..........................................................467 

Periodic Table of the Elements ........................................................................................... 470 

Commonly Used Abbreviations .......................................................................................... 471 


Bacterial Strain Genotypes 

C600 (6) ATCC 23724 F – e14 – (McrA – ) thi-1 thr-1 leuB6 lacY1 fhuA21 glnV44 rfbD1 

C600hfl (1) F – e14 – (McrA – ) thi-1 thr-1 leuB6 lacY1 fhuA21 glnV44 rfbD1 hflA150::Tn10 (Tet r ) hsdR? mcrB? 

DH1 (6) ATCC 33849 F – gyrA96 (Nal r ) recA1 relA1 endA1 thi-1 hsdR17 (r k – mk + ) glnV44 

DH5 (5) F – gyrA96 (Nal r ) recA1 relA1 endA1 thi-1 hsdR17 (r k – mk + ) glnV44 deoR (lacZYA-argF)U169 [80d(lacZ)M15] 

DH5F’ (5) F’/ gyrA96 (Nal r ) recA1 relA1 endA1 thi-1 hsdR17 (r k – mk + ) glnV44 deoR (lacZYA-argF)U169 [80d(lacZ)M15] 

ER1727 (8) F’ proA + B + lacI q (lacZ)M15 / fhuA2 (lacZ)r1 glnV44 trp-31 mcrA1272::Tn10 (Tet r ) his-1 rpsL104 (Str r ) xyl-7 mtl-2 

metB1 (mcrC-mrr)102::Tn10 (Tet r ) 

GM31 (7) F – dcm-6 thr-1 araC14 leuB6 fhuA31 lacY1 tsx-78 glnV44 galK2 galT22 hisG4 rpsL136 (Str r ) xylA5 mtl-1 thi-1 

GM119 (7) ATCC 53339 F – dam-3 dcm-6 lacY1 galK2 galT22 tsx-78 glnV44 metB1 thi? fhuA? mtl? 

GM2163 (7) F – dam-13::Tn9 (Cam r ) dcm-6 hsdR2 (r k – mk + ) leuB6 hisG4 thi-1 araC14 lacY1 galK2 galT22 xylA5 mtl-1 rpsL136 (Str r ) 

fhuA31 tsx-78 glnV44 mcrA mcrB1 

HB101 (6) ATCC 33694 F – leuB6 proA2 recA13 thi-1 araC14 lacY1 galK2 xyl-5 mtl-1 rpsL20 (Str r ) glnV44 (mcrC-mrr) 

JM83 (6) ATCC 35607 F – ara (lac-proAB) rpsL (Str r ) thi [80d(lacZ)M15] 

JM101 (6) ATCC 33876 F’ traD36 proA + B + lacI q (lacZ)M15 / (lac-proAB) thi-1 supE 

JM103 (2) ATCC 39403 F’ traD36 proA + B + lacI q (lacZ)M15 / (lac-pro) thi-1 supE endA1 sbcB15 rpsL (Str r ) hsdR (r k – mk + ) 

JM105 (6) ATCC 47016 F’ traD36 proA + B + lacI q (lacZ)M15 / (lac-pro) thi-1 endA1 sbcB15 rpsL (Str r ) hsdR4 (r k – mk + ) 

JM107 (6) ATCC 47014 F’ traD36 proA + B + lacI q (lacZ)M15 / e14 – (McrA – ) (lac-pro) endA1 gyrA96 (Nal r ) thi-1 hsdR17 (r k – mk + ) glnV44 relA1 

JM108 (3) F – e14 – (McrA – ) (lac-pro) endA1 gyrA96 (Nal r ) thi-1 hsdR17 (r k – mk + ) glnV44 relA1 recA1 

JM109 (6) ATCC 53323 F’ traD36 proA + B + lacI q (lacZ)M15 / e14 – (McrA – ) (lac-proAB) endA1 gyrA96 (Nal r ) thi-1 hsdR17 (r k – mk + ) glnV44 

relA1 recA1 

JM110 (6) ATCC 47013 F’ traD36 proA + B + lacI q (lacZ)M15 / rpsL (Str r ) thr leu thi lacY galK galT ara fhuA tsx dam dcm glnV44 

(lac-proAB) hsdR17 

LE392 (6) ATCC 33572 F – e14 – (McrA – ) hsdR514 (r k – mk + ) glnV44 supF58 lacY1 or lac(I-Y)6 galK2 galT22 metB1 trpR55 

K802 (6) ATCC 33526 F – e14 – (McrA – ) metB1 lacY1 or lac(I-Y)6 galK2 galT22 glnV44 hsdR2 (r k – mk + ) mcrB 

MB408 (3) F – thr-44 araC14 leuB6 (gpt-proA)62 recF143 recB21 recC22 sbcB15 hflA hflB0 hsdR (r k – mk + ) lacY1 sbcC201 tsx-33 

qsr’-0 glnV44 galK2 hisG4 rfbD1 mgl-51 rpsL31 (Str r ) kdgK51 xylA5 mtl-1 tna300::Tn10 (Tet r ) argE3 thi-1 

NM522 (6) ATCC 47000 F’ proA + B + lacI q (lacZ)M15 / (hsdMS-mcrB)5 (r k – mk – ) (lac-proAB) supE thi-1 

NM538 (6) ATCC 35638 F – supF hsdR (r k – mk – ) mcrB 

NM539 (6) ATCC 35639 F – supF hsdR (r k – mk – ) mcrB [P2cox3] 

RR1 (6) ATCC 31343 F – leuB6 proA2 thi-1 araC14 lacY1 galK2 xyl-5 mtl-1 rpsL20 (Str r ) glnV44 (mcrC-mrr) 

1776 (6) ATCC 31244 F – fhuA53 dapD8 minA1 glnV42 (gal-uvrB)40 minB2 rfb-2 gyrA25 (Nal r ) thyA142 oms-2 oms-1 (bioH-asd)29 cycB2 

cycA1 hsdR2 (r k – mk – ) metC65? 

XL1-Blue (4) F’::Tn10 (Tet r ) proA + B + lacI q (lacZ)M15 / recA1 endA1 gyrA96 (Nal r ) thi-1 hsdR17 (r k – mk – ) glnV44 relA1 lac 

Y1088 (6) ATCC 37195 F – (lacZYA-argF)U169 proC::Tn5 (Kan r ) supE supF metB trpR hsdR (r k – mk – ) mcrA rpsL (Str r ) fhuA21 [pMC9 lacI q 

(Tet r Amp r )] 

Y1089 (6) ATCC 37196 F – (Ion) (lacZYA-argF)U169 araD139 rpsL (Str r ) hflA150::Tn10 (Tet r ) [pMC9 lacI q (Tet r Amp r )] 

Y1090 (6) ATCC 37197 F – (Ion) trpC22::Tn10 (Tet r ) supF (lacZYA-argF)U169 araD139 mcrA rpsL (Str r ) [pMC9 lacI q (Tet r Amp r )] 

References 

1. Jendrisak, J., et al., Guide to Molecular Cloning Techniques, Academic Press, 359-371, 1987. 

2. Hanahan, D., J. Mol. Biol., 166, 557-580, 1983. 

3. Schatz, D., et al., Cell, 59, 1035, 1989. 

4. Bullock, W.O., et al., BioTechniques, 5, 376-378, 1987. 

5. Woodcock, D.M., et al., Nucleic Acids Res., 17, 3469-3478, 1989. 

6. ATCC Bacteria and Bacteriophages, 19th edition, 1996. 

7. Palmer, B.R., Marinus, M.G., The dam and dcm strains of Escherichia coli – a review, Gene, 143, 7-8, 1994. 

8. Dila, D., et al., J. Bacteriol, 172, 4888-4900, 1990. 


430 

Genetic Markers 

araC14 Mutation in a regulatory gene of the ara operon. Blocks arabinose catabolism. 

deoR Regulatory gene for the deo operon. Allows uptake of large plasmids. 

endA1 Mutation in the endonuclease I gene. Improves the quality of isolated plasmid DNA. 

F – Strain does not contain an F episome. 

F’ Strain contains an F episome. 

galK Mutation in the galactokinase gene. Blocks catabolism of galactose. 

glnV see supE 

gyrA96 Mutation in the DNA gyrase gene. Confers resistance to nalidixic acid. 

hflA150 Mutation leads to greatly enhanced frequency of lysogenization with phages containing a wild type repressor (cl) gene. 

lacI q Mutation leads to high levels of the lac repressor protein, thus inhibiting transcription from the lac promoter. 

lacY Mutation in the galactoside permease gene. Blocks lactose utilization. 

lacZM15, (lacZ)M15 Partial deletion of -galactosidase gene. Allows blue/white selection for recombinant colonies when plated on X-Gal/IPTG. 

leuB Mutation in -isopropyl malate dehydrogenase gene. Strains require leucine for growth on minimal media. 

(lon) Deletion of lon protease gene. Reduces proteolysis of expressed fusion proteins. 

metB Mutation in cystathionine -synthase gene. Strains require methionine for growth on minimal media. 

mtl1 Mutation in mannitol metabolism genes. Blocks catabolism of mannitol. 

P1 P1 bacteriophage 

proAB Mutation in proline metabolism genes. Strains require proline for growth on minimal media. 

recA1, recA13 Mutation in general recombination gene. 

recB, recC Mutation in exonuclease V gene. Reduces general recombination and affects repair of radiation damage. 

relA Relaxed phenotype, mutation eliminating stringent factor. Allows RNA synthesis in the absence of protein synthesis. 

rpsL Mutation in subunit S12 of 30S ribosome gene. Confers resistance to streptomycin. 

sbcB Mutation in exonuclease I gene. Allows general recombination in recBC mutant strains. 

supE Suppressor of amber (UAG) mutation. Now called glnV. 

supF Suppressor of amber (UAG) mutation. Now called tyrT. 

thi1 Mutation in thiamine metabolism gene. Strains require thiamine for growth on minimal media. 

Tn5 Transposon conferring resistance to kanamycin. 

Tn10 Transposon conferring resistance to tetracycline. 

tonA Mutation in outer membrane protein gene. Confers resistance to bacteriophage T1. Now called fhuA. 

traD36 Mutation in transfer factor gene. Prevents transfer of F episome. 

tyrT see supF 

xyl5 Mutation in xylose metabolism gene. Blocks catabolism of xylose. 


For less common genetic markers, see Bachmann, B.J., Microbiological Reviews, 54, 130-197, 1990.

E.coli Host Restriction and Modification Systems 

Table 12.1. E.coli Host restriction and modification systems 

System Description Effects on cloning experiments 

EcoKI, EcoBI 

Dam 

Dcm 

McrA 

McrBC 

Mrr 

Type I restriction-modification systems are encoded by host hsdR, 

hsdM and hsdS genes (1). Products of all three genes form a multisubunit 

enzyme possessing both endonucleolytic and methylation 

activities (1). The enzyme methylates the unmodified DNA strand 

when the recognition sequence is hemimethylated. When the 

recognition site is completely unmodified, the enzyme hydrolyzes 

DNA as an endonuclease. In addition, products of hsdM and hsdS 

form a complex capable of modifying specific DNA targets. EcoKI 

recognizes the sequence 5’...AAC(N) 6GTGC (2), EcoBI recognizes 

the sequence 5’...TGA(N) 8TGCT (3). 

DNA adenine methylase is encoded by the dam gene of E.coli. It 

modifies the adenine residue at the N6-position on both strands 

within the sequence 5’...GATC (4, 5). 

DNA cytosine methylase is encoded by the dcm gene of E.coli. It 

modifies the internal cytosine residue at the C5-position on both 

strands within the sequence 5’...CC(A/T)GG (9, 10). 

The E.coli chromosome-encoded restriction system named McrA 

(for Modified cytosine restriction) is directed against DNA containing 

either 5-hydroxymethylcytosine or 5-methylcytosine. At present, 

only the T-even phages and DNA modified at HpaII sites 5’... 

Cm5CGG are known targets for McrA (11, 12). 

McrBC is the other chromosome-encoded E.coli restriction system, 

which, like McrA, is specific for modified cytosine. It is encoded 

by two neighbor genes, mcrB and mcrC (14). McrBC requires 

the presence of two (G/A)mC recognition elements (where mC is 

5-hydroxymethylcytosine, N4-methylcytosine or 5-methylcytosine 

(14)) appropriately spaced in the substrate DNA (15). DNA cleavage 

occurs in the region between two recognition elements (15). The 

optimal separation between elements is 55-103 base pairs (15). 

The E.coli chromosome-encoded restriction system named Mrr 

(for Methylated adenine recognition and restriction) targets DNA 

containing both N6-methyladenine and 5-methylcytosine (16-18). 

No simple consensus sequence causing Mrr-associated restriction 

has been determined. 

References 

1. Yuan, R., , et al. (eds.), DNA methylation, Biochemistry and Biological Significance, 

Springer-Verlag, New York, 11-38, 1984. 

2. Kan, N.C., et al., J. Mol. Biol., 130, 191-209, 1979. 

3. Lautenberger, J.A., et al., Proc. Natl. Acad. Sci. USA, 75, 2271-2275, 1978. 

4. Hattman, S., et al., J. Mol. Biol., 126, 367-380, 1978. 

5. Geier, G.E., Modrich, P., J. Biol. Chem., 254, 1408-1413, 1979. 

6. Messer, W., et al., EMBO J., 4, 1327-1332, 1985. 

7. Smith, D.W., et al., EMBO J., 4, 1319-1326, 1985. 

8. Russell, D.W., Zinder, N.D., Cell, 50, 1071-1079, 1987. 

The majority of laboratory strains of Escherichia coli have 

been derived from wild-type strains K-12 and B, which 

possess the EcoKI and EcoBI restriction-modification 

systems, respectively. hsdS mutations abolish both DNA 

modification and restriction; hsdR mutants are modificationproficient, 

but they are restriction-deficient. Therefore, it is 

difficult to transform wild-type E.coli (hsdR + M + S + ) using a 

plasmid containing EcoKI and EcoBI recognition sites isolated 

from the hsdM, hsdS mutants of E.coli. These sites remain 

unprotected by methylation in such a plasmid. This results 

in plasmid destruction in wild-type cells. Modification of the 

EcoKI site or the EcoBI site may reduce DNA cleavage by 

restriction endonucleases whose targets overlap 

with these sites. 

E.coli 

chromosomal replication origin or the pMB1 plasmid 

origin transform poorly the dam mutants of E.coli (6, 

7). But these mutants are transformed efficiently with 

unmethylated DNA (8), while both methylated and 

unmethylated plasmids transform wild-type dam + strains 

efficiently. 

E.coli dam + strains can be difficult to 

hydrolyze by restriction endonucleases whose targets 

overlap (partially or completely) with the Dam recognition 

site (see p.174). 

DNA isolated from E.coli dcm + strain can be difficult to 

hydrolyze by restriction endonucleases whose targets overlap 

(partially or completely) with the Dcm recognition site (see 

p.175). 

The McrA + phenotype of the host can interfere with the 

cloning of appropriately modified DNA (or genes coding for 

DNA modification enzymes of either CCGG or overlapping 

specificities). McrA phenotypes of some laboratory E.coli 

strains are presented in reference 13. 

The McrBC + phenotype of the host can interfere with the 

cloning of appropriately modified DNA or genes coding for 

DNA modification enzymes with overlapping specificity. 

McrBC phenotypes of some laboratory E.coli strains are 

presented in reference 13. 

The precise specificity of Mrr is uncertain, but it requires 

the presence of either modified adenine or cytosine. Thus, 

the Mrr + phenotype of the host can interfere with cloning of 

appropriately modified DNA, as well as genes coding for DNA 

modification enzymes with overlapping specificity. 

9. Buryanov, Y.I., et al., FEBS Lett., 88, 251-254, 1978. 

10. May, M.S., Hattman, S., J. Bacteriol., 123, 768-770, 1975. 

11. Raleigh, E., Wilson, G., Proc. Natl. Acad. Sci. USA, 83, 9070-9074, 1986. 

12. Raleigh, E., et al. J. Bacteriol., 173, 2707-2709, 1991. 

13. Raleigh, E., et al., Nucleic Acids Res., 16, 1563-1575, 1988. 

14. Dila, D., et al., J. Bacteriol., 172, 4888-4900, 1990. 

15.Stewart, F.J., Raleigh, E.A., Biol. Chem., 379, 611-616, 1998. 

16. Heitman, J., Model, P., J. Bacteriol., 169, 3243-3250, 1987. 

17. Kelleher, J.E., Raleigh, E.A., J. Bacteriol., 173, 5220-5223, 1991. 

18. Waite-Rees, P.A., et al., J. Bacteriol., 173, 5207-5219, 1991. 


432 

Phage and Plasmid DNA Maps 

pJET1.2 

Available with the CloneJET PCR Cloning Kit, see p.279 

The positive selection vector pJET1.2 is a small, high 

copy number E.coli plasmid, 2974 bp in length. It was 

derived from pUC19 by replacing the 5’-terminal part 

of the lacZ gene encoding the N-terminal fragment 

of -galactosidase with the gene coding for the 

Eco47I restriction endonuclease. In the absence of 

cognate methylation, the endonuclease is lethal to 

E.coli host cells. Positive selection with the pJET1.2 

plasmid is mediated by insertional inactivation of the 

eco47I gene. For convenience, the multiple cloning 

site (MCS) used for positive selection, as well as the 

T7 polymerase promoter, were incorporated into 

eco47IR by silent mutagenesis. The pJET1.2 plasmid 

contains: (1) the pMB1 replicon rep responsible for 

the replication of plasmid (source – plasmid pUC19). 

The high copy number of pJET1.2 is a result of the 

lack of the rop gene and a single point mutation in 

the replicon rep of pMB1; (2) the bla gene, coding 

for -lactamase, that confers resistance to ampicillin 

(source – plasmid pUC19); (3) the region of E.coli 

lac operon containing a CAP protein binding site, 

Multiple Cloning Site 


promoter P lacUV5 which differs from the wild-type 

promoter P lac by two point mutations in -10 region, 

and lac repressor binding site which contains 

two mutations resulting in reduced binding of the 

repressor (source – plasmid pUC19; mutations were 

introduced by site-specific mutagenesis); (4) the 

modified eco47IR gene, coding for Eco47I restriction 

endonuclease. The gene is toxic for all E.coli strains 

that are not protected by cognate methylation. The 

background transcription from P lacUV5 ensures the 

efficient killing of recipient cells even in cases where 

the stain contains the lacI q mutation which ensures 

elevated intracellular concentration of LacI repressor. 

Therefore, the induction of Eco47I synthesis by IPTG 

is not required for positive selection purposes. After 

the cloning of DNA into eco47IR the integrity of this 

gene is disrupted. Therefore, only those cells that 

contain the recombinant plasmid are able to survive 

and form a colony in presence of ampicillin. 

The plasmid map provided below lists enzymes which 

have unique targets within pJET1.2. The coordinates 

refer to the position of the first nucleotide in each 

recognition sequence. 

The exact positions of the genetic elements are shown 

on the map (termination codons included). The bla 

gene nucleotides 2782-2714 (complementary strand) 

code for a signal peptide. The indicated rep region 

is sufficient to promote replication. DNA replication 

initiates at position 1162 (+/-1) and proceeds in the 

direction indicated. Plasmids carrying the pMB1 and 

ColE1 replicons are incompatible, but they are fully 

compatible with those carrying the p15A replicon 

(pACYC177, pACYC184). pMB1-derived plasmids can 

be amplified using chloramphenicol. 

GenBank/EMBL Accession Number 

EF694056 

Additional Information 

 

strand). 

Enzymes which cut pJET1.2 DNA once: 

DrdI 1204 

AdeI 120 

AloI 284 

BbvCI 717 

BceAI 1588 

BcgI 2511 

BmrI 2040 

BglI 2109 

BpiI 58 

Bpu10I 717 

BsaXI 955 

Bsu15I* 417 

BtgI* 408 

BveI 219 

CaiI 1513 

Cfr10I 2075 

Csp6I 2474 

Eam1105I 1990 

Eco130I* 408 

Eco31I 2062 

Eco32I* 369 

Eco52I* 329 

Eco88I* 352 

Esp3I 739 

FaqI 103 

GsuI 2080 

HindIII 624 

Kpn2I* 343 

LguI 979 

MssI 761 

MunI 892 

Mva1269I 722 

NcoI* 408 

NmeAIII 2118 

NotI* 328 

NsbI 2215 

PdmI 2590 

PspFI 1406 

PspXI* 351 

PstI 5 

PvuI 2362 

RsaI 2474 

ScaI 2473 

FauI 951 

TatI 2473 

XbaI* 377 

XhoI* 

* – MCS 

352 

pJET1.2 forward sequencing primer, 23-mer Eco88I 

Eco52I XhoI 

T7 promoter T7 transcription start 328 NotI BglII Kpn2I PspXI 

5’ - GGC GTA ATA CGA CTC ACT ATA GGG AGA GCG GCC GCC AGA TCT TCC GGA TGG CTC GAG TTT TTC AGC 

3’ - CCG CAT TAT GCT GAG TGA TAT CCC TCT CGC CGG CGG TCT AGA AGG CCT ACC GAG CTC AAA AAG TCG 

Ala Tyr Tyr Ser Glu Ser Tyr Pro Ser Arg Gly Gly Ser Arg Gly Ser Pro Glu Leu Lys Glu Ala 

BtgI 

Eco130I 

Eco32I XbaI BglII NcoI Bsu15I 422 

AAG ATA TCT TTC TAG AAG ATG TCC TAC AAT ATT CTC AGC TGC CAT GGA AAA TCG ATG TTC TTC T - 3’ 

TTC TAT AGA AAG ATC TTC TAG AGG ATG TTA TAA GAG TCG ACG GTA CCT TTT AGC TAC AAG AAG A - 5’ 

Leu Tyr Arg Glu Leu Leu Asp Gly Val Ile Asn Glu Ala Ala Met Ser Phe Arg His Glu Glu 

pJET1.2 reverse sequencing primer, 24-mer

Table 12.2. pJET1.2 DNA Restriction Sites 

Enzyme 

1 2 3 

Number of recognition sites 

4 5 6 7 8 9 10 11 12 

DrdI 1204 

AatII 737 2913 

AdeI 120 

AflIII 750 1102 

AloI 284 

Alw21I 1416 2577 2662 

Alw26I 639 740 2062 2827 

Alw44I 1416 2662 

BauI 1275 2659 2966 

BbvCI 717 

BccI 295 347 2031 2155 2442 

BceAI 1588 

BcgI 2511 

BcnI 1481 2177 2528 

BmrI 2040 

BfmI 5 317 1367 1558 2236 

BfuI 1311 2838 

BglI 2109 

BglII 337 383 

Bme1390I 838 1129 1250 1263 1481 2177 2528 

BpiI 58 

Bpu10I 717 

BpuEI 439 1208 1470 1747 2615 

BsaWI 343 1308 1455 2286 

BsaXI 955 

BseDI 408 838 1262 

BseLI 944 1118 1136 1302 1581 

BseMI 2049 2231 

BseMII 400 637 718 1377 1786 1952 2492 

BseNI 902 1505 1518 1635 2041 2159 2202 2466 2641 

BseSI 1416 2662 

BseXI 7 404 926 1007 1025 1444 1509 1512 1718 2046 2412 

Bsh1236I 948 950 1148 1729 2059 2552 2884 

Bsh1285I 329 1015 1439 2362 2511 

BshNI 843 1943 

BspLI 222 500 843 1132 1171 1943 2037 2078 2289 2879 

BspPI 1669 1743 1755 1840 1853 2317 2620 2638 

Bsu15I 417 

BsuRI 330 942 1116 1127 1145 1579 2037 2117 2384 2971 

BtgI 408 

BtsI 886 2388 2416 

BveI 219 

Cac8I 622 922 1031 1117 1154 1714 2105 

CaiI 1513 

EaeI 329 941 2383 

Cfr10I 2075 

Cfr13I 2037 2116 2133 2355 

CseI 1204 1782 2532 

Csp6I 2474 

DraI 68 661 755 762 1859 1878 2570 

Eam1104I 980 2784 

Eam1105I 1990 

EciI 1174 1320 2148 

Eco31I 2062 

Eco32I 369 

Eco47I 2133 2355 

Eco52I 329 

Eco57I 53 627 1629 2677 

Eco88I 352 

Eco130I 408 

EcoP15I 1510 1626 1719 

EcoRII 838 1129 1250 1263 

Esp3I 739 

FaqI 103 

FokI 296 346 693 1975 2156 2443 

FspBI 378 1597 1850 2185 

GsuI 2080 

There are no restriction sites in pJET1.2 DNA for the following enzymes: 

AanI, AarI, AbsI, Acc65I, AjiI, AjuI, AlfI, ApaI, BaeI, BamHI, BclI, BcuI, BoxI, 

BplI, Bpu1102I, BseJI, BseRI, BsgI, BshTI, Bsp119I, Bsp120I, Bsp1407I, 

Bsp68I, BspOI, BspTI, Bst1107I, BstAPI, BstXI, BtgZI, Cfr42I, Cfr9I, CpoI, 

CspCI, Ecl136II, Eco105I, Eco147I, Eco24I, Eco47III, Eco72I, Eco81I, Eco91I, 

EcoO109I, EcoRI, EheI, FalI, FseI, FspAI, HincII, KpnI, KspAI, MauBI, MlsI, 

MluI, Mph1103I, MreI, NdeI, NheI, OliI, PacI, PaeI, PasI, PauI, PdiI, Pfl23II, 

PfoI, Ppu21I, Psp5II, PsrI, PsyI, SacI, SalI, SanDI, SdaI, SexAI, SfaAI, SfiI, 

SgrAI, SgrDI, SgsI, SmaI, SrfI, SspDI, Van91I, XagI, XcmI, XmaJI, XmiI. 

Enzyme 

1 2 3 


4 5 6 7 8 9 10 11 12 

HaeII 976 1346 

Hin1I 737 2531 2913 

Hin1II 409 543 751 1103 1823 2314 2324 2402 2438 2831 2936 

HindIII 624 

HinfI 311 537 937 1002 1077 1473 1990 

HpaII 299 344 817 1309 1456 1482 1672 2076 2110 2177 2287 2529 

HphI 1846 2073 2469 2695 2710 

Hpy8I 1416 1904 2662 

Hpy99I 1203 1997 2260 

Hpy188I 52 1214 1292 1645 1779 1914 2360 2371 2491 

HpyAV 1332 1600 1642 2113 2350 2725 

HpyCH4V 6 596 709 928 1417 1713 2048 2138 2331 2419 2663 

HpyF3I 76 400 637 718 1377 1786 1952 2492 2918 

HpyF10VI 1 837 881 968 1035 1149 1721 2109 2972 

Kpn2I 343 

LguI 979 

Lsp1109I 7 404 926 1007 1025 1444 1509 1512 1718 2046 2412 

LweI 116 692 1190 2242 2452 2682 

MaeIII 1459 1522 1638 1921 2252 2310 2463 2651 

MbiI 326 789 1033 2834 

MboII 59 62 340 381 425 671 981 1752 1843 2598 2676 2785 

MmeI 1292 1476 

MspA1I 402 924 1442 1687 2628 

MspI 299 344 817 1309 1456 1482 1672 2076 2110 2177 2287 2529 

MssI 761 

MunI 892 

MvaI 838 1129 1250 1263 

Mva1269I 722 

NcoI 408 

NmeAIII 2118 

NmuCI 2252 2463 

NotI 328 

NsbI 2215 

PagI 542 1822 2830 2935 

PdmI 2590 

PfeI 937 1077 

PscI 750 1102 

Psp1406I 2220 2593 

PspFI 1406 

PspXI 351 

PstI 5 

PsuI 337 383 1743 1754 1840 1852 2620 2637 

PvuI 2362 

PvuII 402 924 

RsaI 2474 

RseI 243 2243 2402 2761 

ScaI 2473 

SchI 311 537 1002 1473 1990 

SduI 1416 2577 2662 

SmiI 67 660 

SmoI 352 439 1208 1470 1747 2615 

FauI 951 

SspI 394 561 2797 

TaaI 503 1064 1135 1605 1918 2433 

TaiI 738 1805 2221 2594 2914 

TaqI 180 353 418 535 1202 2646 

TatI 2473 

TauI 328 331 1028 1146 1301 2385 2507 2736 2972 

TscAI 886 998 1504 1517 1788 1937 2042 2389 2416 

TseI 7 404 926 1007 1025 1444 1509 1512 1718 2046 2412 

TspDTI 64 88 229 241 544 935 1871 1973 2276 

TspGWI 2445 2762 

VspI 869 931 2166 

XapI 71 646 658 664 

XbaI 377 

XceI 750 1102 

XhoI 352 

Indicates restriction sites of Thermo Scientific enzymes that are sensitive (cleavage completely 

blocked or partially inhibited) to overlapping Dam/Dcm methylation. Complete cleavage is achieved with DNA 

substrates isolated from dam – or dcm – hosts. 


434 

Phage Lambda 

see p.291 for ordering information. 

is a temperate Escherichia coli bacteriophage. 

The virion DNA is linear and double-stranded 

(48502 nt) with 12 bp single-stranded 

complementary 5’-ends. After the phage 

particle injects its chromosome into the cell, 

the chromosome circularizes by end joining. 

In the lytic pathway, phage genes encoding 

replication, lysis and virion proteins are 

expressed. The chromosome replicates and the 

replicas are cleaved and packaged into progeny 

phage particles. In the lysogenic pathway, 

ApaI 

Bsp120I 


Eco105I 

Pfl23II PdiI 

phage gene expression is repressed, and the 

circular chromosome inserts into the bacterial 

chromosome by recombination. 

Phage DNA is a common substrate for 

restriction endonucleases and for generating DNA 

size marker fragments. For large scale isolation 

of phage DNA, cI857Sam7, a mutant carrying 

four known mutations, is often used. The DNA 

sequence used to construct a phage restriction 

map includes these mutations. The map shows 

enzymes that cut DNA once. Thermo Scientific 

enzymes are shown in orange. 

SanDI 

PspXI BspOI 

XhoI NheI 

0 10,000 20,000 30,000 SgrDI 40,000 

Table 12.3. DNA Restriction Sites 

XbaI 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

AanI 2285 9011 18941 19573 22014 24667 25472 27746 29498 29656 33236 37756 

AarI 554 10835 13941 14383 16164 16194 16233 21242 29897 36293 37003 38481 

DrdI 5116 9104 11090 

AatII 5105 9394 11243 14974 29036 40806 41113 42247 45563 45592 

Acc65I 17053 18556 

AdeI 2954 5613 6635 8999 14477 30365 31909 41479 47312 48434 

AjuI 37231 40534 43659 

AloI 266 7286 12056 21639 24654 36718 42867 

Alw44I 5619 21798 27173 40216 

ApaI 10086 

BaeI 694 7665 13267 15775 16271 18732 19109 21779 43976 48385 

BamHI 5505 22346 27972 34499 41732 

BauI 20356 25572 27956 29425 34430 35219 42416 42737 

BbvCI 8012 18147 18465 30916 31222 31836 35813 

BclI 8844 9361 13820 32729 37352 43682 46366 47942 

BmrI 7054 11608 25691 30332 

BglII 415 22425 35711 38103 38754 38814 

BoxI 8920 9394 13512 15412 36925 37889 48152 

BplI 41036 

Bpu1102I 10297 10682 11661 16518 20744 39450 

BseSI 5619 5664 10086 11414 13039 14897 21798 27173 32330 40216 

Bsp119I 18048 25884 27980 29150 30396 34331 42637 

Bsp120I 10086 

Bsp1407I 5220 6142 15855 29392 32496 

Bsp68I 4590 28050 31703 32407 41808 

BspOI 34679 

BspTI 6540 12618 42630 

Bst1107I 15260 18834 19473 

Cfr9I 19397 31617 39888 

Cfr42I 20320 20530 21606 40386 

CpoI 3800 6041 13983 19288 22242 

CspCI 10780 10800 15063 33276 41481 46577 48429 

Eam1105I 6398 11238 12477 12915 16588 18544 23460 30467 44669 

Ecl136II 24772 25877 

Eco24I 581 10086 19763 21570 24772 25877 39453 

Eco31I 11424 42715 

Eco47III 20995 37057 

Eco52I 19944 36654 

Eco72I 26529 41482 42362 

Eco81I 26717 34318 

Eco88I 4720 19397 20999 27887 31617 33498 38214 39888 

Eco105I 12188 

Eco130I 19329 21211 23901 24322 24396 27868 28793 35016 36505 44248 

Eco147I 12434 31478 32997 39992 40596 40614 

EcoO109I 2815 28797 48473 

EcoRI 21226 26104 31747 39168 44972 

EheI 45679 

FspAI 21804 21825 

HindIII 23130 25157 27479 36895 37459 44141 

KpnI 17053 18556 

LguI 2397 6489 8702 10370 13286 24769 27234 34327 34800 47712 

MluI 458 5548 15372 17791 19996 20952 22220 

MssI 8459 16293 

MunI 22687 22715 23054 25863 35764 37186 38332 47880 

NcoI 19329 23901 27868 44248 

BplI 

SspDI 

EheI 

GenBank/EMBL Accession Numbers 

J02459, M17233, M24325, V00636, X00906. 

Enzymes which cut DNA once: 

ApaI 10086 

BplI 41036 

Bsp120I 10086 

BspOI 34679 

Eco105I 12188 

EheI 45679 

NheI 34679 

PdiI 20040 

Pfl23II 19323 

PspXI 33497 

SanDI 28797 

SgrDI 33243 

XbaI 24508 

XhoI 33498 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

NdeI 27630 29883 33679 36112 36668 38357 40131 

NheI 34679 

NmeAIII 1041 3190 16533 17983 20435 31939 32283 38107 

PaeI 2212 12002 23942 24371 27374 39418 

PagI 889 4650 4989 10249 18275 28860 31608 40642 

PasI 15323 19188 

PauI 3522 4126 5627 14815 16649 28008 

PdiI 20040 

Pfl23II 19323 

PscI 628 39395 

Psp1406I 13529 16290 22580 22595 24642 43392 43488 

Psp5II 2815 28797 48473 

PspXI 33497 

PsrI 1697 20889 33098 

PsyI 11202 36120 

PvuI 11933 26254 35787 

SacI 24772 25877 

SalI 32745 33244 

SanDI 28797 

ScaI 16421 18684 25685 27263 32802 

SdaI 2555 2819 11834 19832 37000 

SexAI 22264 31009 32838 40497 44408 

SgrAI 7064 8680 12878 15653 16974 31824 

SgrDI 33243 

SgsI 3521 16648 

SmaI 19397 31617 39888 

SspDI 45679 

XagI 13509 21292 22377 25174 25223 35521 38268 41842 47213 

XbaI 24508 

XcmI 958 4770 5065 5874 9193 9460 9490 14891 19329 23685 32530 36165 

XhoI 33498 

XmaJI 24322 24396 

XmiI 2190 15260 18834 19473 31301 32745 33244 40201 42921 

Indicates restriction sites of Thermo Scientific enzymes that are sensitive (cleavage completely blocked 

or partially inhibited) to overlapping Dam/Dcm methylation. Complete cleavage is achieved with DNA substrates 

isolated from dam – or dcm – hosts. 

There are no restriction sites in DNA for the following enzymes: 

AbsI, BcuI, FseI, MauBI, MreI, NotI, PacI, SfaAI, SfiI, SmiI, SrfI. 

References 

1. Daniels, D.L., et al., Appendix I: A molecular map of coliphage lambda. (in) Hendrix,R.W., 

Roberts,J.W., Stahl,F.W. and Weisberg,R.A. (Eds.), LAMBDA II: 469-517, Cold Spring Harbor 

Laboratory, Cold Spring Harbor, 1983. 

2. Daniels, D.L., et al., Appendix II: Complete annotated lambda sequence. (in) Hendrix, R.W., 

Roberts, J.W., Stahl, F.W. and Weisberg, R.A. (Eds.), LAMBDA II: 519-674, Cold Spring Harbor 

Laboratory, Cold Spring Harbor, 1983. 

3. Sanger, F., et al., Nucleotide sequence of bacteriophage lambda DNA, J. Mol. Biol. ,162, 

729-773, 1982.

X174 


X174 was the first DNA virus discovered 

to have a single-stranded, circular genome. 

X174 DNA is 5386 nt in length. The DNA 

strand packaged into the virion is termed the 

“plus” strand. After entering the cell, X174 

DNA is used as a template for minus-strand 

synthesis, producing double-stranded DNA. The 

conversion of plus DNA strands to doublestranded 

DNA does not require any of the phage 

genes to function. The double-stranded DNA 

can then be transcribed, resulting in synthesis 

of phage-encoded proteins. Synthesis of single- 

Coordinates of X174 genes 

(termination codons included): 

A 3981-136 

A* 4497-136 

B 5075-51 

C 133-393 

D 390-848 

E 568-843 

F 1001-2284 

G 2395-2922 

H 2931-3917 

J 848-964 

K 51-221 

References 

1. Air, G.M., et al., Nucleotide sequence of the F protein 

coding region of bacteriophage X174 and the amino acid 

sequence of its product, J. Mol. Biol., 125, 247-254, 1978. 

2. Sanger, F., et al., Nucleotide sequence of bacteriophage 

X174 DNA, Nature, 265, 687-695, 1977. 

3. Sanger, F., et al., The nucleotide sequence of bacteriophage 

X174, J. Mol. Biol., 125, 225-246, 1978. 

stranded (plus) DNA requires phage-encoded 

gene A protein. DNA synthesis is initiated at 

ori (+) and proceeds in the direction indicated. 

Late in infection, the single-stranded circles 

are encapsidated into new virions. The cycle 

terminates by cellular lysis, mediated by phage 

gene E encoded protein. 

The genes identified in phage X174 are 

shown on the map. All genes are transcribed 

clockwise. Enumeration of phage DNA begins 

with the last nucleotide of the unique PstI site 

and continues clockwise around the viral (+) 

strand in the 5’3’ direction. The map shows 

enzymes that cut X174 DNA once. Thermo 

Scientific enzymes are shown in orange. The 

coordinates refer to the position of the first 

nucleotide in each recognition sequence. 


V01128, J02482, M10348, M10379, M10714, 

M10749, M10750, M10866, M10867, 

M24859. 

Enzymes which cut X174 DNA once: 

AanI 2304 

DrdI 5171 

AatII 2782 

AdeI 5183 

AjuI 5113 

Alw44I 4779 

BaeI 2581 

BcnI 2800 

BoxI 1694 

BseSI 4779 

Bsh1285I 4601 

BtsI 2203 

Cfr42I 2859 

Eam1105I 1760 

Eco47I 5042 

Eco88I 162 

Eco147I 4486 

FalI 3172 

LguI 3745 

MbiI 530 

MunI 3939 

NsbI 155 

OliI 2912 

PauI 5348 

PstI 5382 

SexAI 3499 

SspI 1007 

XhoI 162 


436 

Table 12.4.X174 DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

AanI 2304 

DrdI 5171 

AatII 2782 

AdeI 5183 

AflIII 221 2146 

AjuI 5113 

Alw21I 538 4290 4779 

Alw26I 673 1529 2903 3441 

Alw44I 4779 

BaeI 2581 

BauI 535 3280 

BbvCI 2039 2341 3699 

BccI 192 1048 1129 1262 1492 1751 2577 2743 3885 4118 4560 4897 

BceAI 462 627 666 895 1136 1414 3210 3642 4204 4759 5228 

BcgI 967 5326 

BcnI 2800 

BfmI 1191 3061 4318 4801 5293 5382 

BfuI 588 2580 3275 5373 

Bme1390I 881 2800 3500 

BoxI 1694 

BpiI 2679 4367 4762 

Bpu10I 1361 1835 2039 2341 2379 3306 3699 

BpuEI 987 3631 

BsaWI 728 1102 3018 

BseDI 880 1415 1434 2859 4681 5182 

BseGI 228 648 1261 1419 1996 4265 4463 5152 

BseJI 341 2716 

BseMI 632 1161 2566 4539 

BseMII 350 1527 1533 1836 2040 2342 3307 3700 4252 4738 

BseNI 272 1016 1301 1777 2176 2185 3404 4850 4984 

BseRI 232 1278 

BseSI 4779 

Bsh1285I 4601 

BshNI 1019 2478 2976 

Bsp68I 2260 4424 

BspLI 978 1019 2119 2478 2688 2976 

BspTI 2914 4413 

BstAPI 892 4781 

BstXI 1777 2739 4984 

BsuRI 434 668 978 1172 1775 3128 4206 4487 4758 4876 4948 

BtgI 1415 2859 5182 

BtgZI 4426 4898 

BtsI 2203 

BveI 3599 4052 5380 

EaeI 4205 4757 

Cfr13I 978 5042 

Cfr42I 2859 

Csp6I 414 571 768 906 1431 1903 2150 2795 3187 4747 4836 

CspCI 1563 4866 

DraI 327 1406 

Eam1104I 3746 4143 

Eam1105I 1760 

EciI 125 1876 

Eco47I 5042 

Eco88I 162 

Eco147I 4486 

There are no restriction sites in X174 DNA for the following enzymes: 

AarI, AbsI, Acc65I, AjiI, AlfI, AloI, ApaI, BamHI, BclI, BcuI, BmrI, BglI, BglII, 

BplI, Bpu1102I, BsaXI, BsgI, BshTI, Bsp119I, Bsp120I, Bsp143I, Bsp1407I, 

BspOI, BspPI, Bst1107I, Bsu15I, CaiI, Cfr9I, Cfr10I, CpoI, DpnI, Ecl136II, 

Eco24I, Eco31I, Eco32I, Eco47III, Eco52I, Eco57I, Eco72I, Eco81I, Eco91I, 

Eco105I, Eco130I, EcoO109I, EcoRI, Esp3I, FseI, FspAI, HindIII, Kpn2I, 

KpnI, MauBI, MboI, MlsI, Mph1103I, MreI, MssI, NcoI, NdeI, NheI, NotI, 

PacI, PaeI, PasI, PdiI, PfoI, PscI, Psp5II, PspXI, PsuI, PsyI, PvuI, PvuII, 

SacI, SalI, SanDI, ScaI, SdaI, SfaAI, SfiI, SgfI, SgrAI, SgsI, SmaI, SmiI, FauI, 

SrfI, TatI, XagI, XbaI, XceI, XcmI, XmaJI. 



Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

EcoP15I 120 3295 3349 3597 4399 

EcoRII 881 3500 

EheI 1019 2976 

FalI 3172 

FaqI 1214 4339 

FokI 228 648 1261 1419 1996 4265 4463 5152 

FspBI 3136 3907 5061 

GsuI 2465 2905 4482 

HaeII 687 872 926 1019 1142 1411 2976 3759 

Hin1I 717 1019 1133 2782 2976 3363 5225 

HpaII 729 1103 2800 3019 3367 

HphI 1235 2012 2089 2132 2923 3033 3072 4188 4965 

Hpy99I 543 719 1166 1767 4830 5219 5224 5300 

KspAI 28 1292 5022 

LguI 3745 

LweI 358 1420 1466 1733 2638 3075 3339 3431 3521 3795 4129 4956 

MbiI 530 

MboII 480 804 1661 2473 2562 2680 2683 3747 4143 4367 4763 

MluI 221 2146 

MmeI 225 2691 3237 5197 5376 

MspA1I 739 2339 2859 3259 3735 

MspI 729 1103 2800 3019 3367 

MunI 3939 

MvaI 881 3500 

Mva1269I 2570 3025 3463 4535 

NmeAIII 1022 3426 4680 

NmuCI 288 1224 2231 2773 3789 4009 4642 5322 

NsbI 155 

OliI 2912 

PagI 388 1254 2271 

PauI 5348 

PdmI 3857 4143 5117 

PfeI 204 310 1897 2015 2264 2877 3377 3731 4148 4701 5128 

Pfl23II 413 2794 

Ppu21I 826 4871 

Psp1406I 864 2514 2809 

PspFI 5210 5279 

PsrI 405 858 

PstI 5382 

RsaI 414 571 768 906 1431 1903 2150 2795 3187 4747 4836 

RseI 1226 2165 2571 2740 2912 4554 4767 

SchI 53 286 352 392 1118 1184 2677 3443 3543 3683 

SduI 538 4290 4779 

SexAI 3499 

SmoI 162 987 2914 3631 4413 

SspI 1007 

SspDI 1019 2976 

TaqI 56 110 143 163 490 721 1125 4039 4180 5355 

TscAI 782 1241 2048 2177 2204 3791 

TspGWI 411 699 1149 1482 2004 2189 5184 

Van91I 3960 4095 

VspI 711 4308 

XapI 139 693 1983 2222 2324 2650 5340 

XhoI 162 

XmiI 1193 3545 



substrates isolated from dam – or dcm – hosts.

M13mp18, M13mp19 

Not available from us 

M13mp18 and M13mp19 vectors are derivatives of 

the single-stranded, male-specific filamentous DNA 

bacteriophage M13. Both vectors are 7250 bp in 

length and have the same MCS inserted in opposing 

orientations. The vector DNAs contain a region of 

E.coli operon lac containing a CAP protein binding 

site, promoter Plac, a lac repressor binding site and 

the 5’-terminal part of the lacZ gene encoding the 

N-terminal fragment of -galactosidase (codons 6-7 

of lacZ are replaced by MCS). This fragment, whose 

synthesis can be induced by IPTG, is capable of 

intra-allelic () complementation with a defective form 

of -galactosidase encoded by the host (mutation 

(lacZ)M15). This results in the appearance of 

blue plaques on media containing IPTG and X-Gal. 

Recombinant phages containing inserts that destroy 

the reading frame of lacZ are revealed as colorless 

plaques. Synthesis of viral (plus) single-stranded DNA 

Multiple Cloning Site of M13mp18 

M13/pUC reverse sequencing primer, 17-mer, (-26) (#S0101) 

CAG GAA ACA GCT ATG ACC ATG ATT 

Met Thr Met Ile 

requires phage-encoded gene II, X and V proteins. 

It is initiated at ori (+) and proceeds in the direction 

indicated. The conversion of plus DNA strands 

to double-stranded DNA does not require any of 

the phage genes. DNA synthesis is initiated by a 

30-nucleotide RNA primer synthesized by the host’s 

RNA polymerase and starts at ori (-). Double-stranded 

circular DNA (replicative form, or RF) can be isolated 

from cells by standard plasmid preparation techniques 

and used for cloning experiments, while the singlestranded 

viral DNA (+ strand) can be isolated from 

phage particles collected from culture medium. 

M13mp18/19 genes are shown on the map (M13 

genes are transcribed clockwise). The map shows 

enzymes that cut M13mp18/19 DNA once. Thermo 


coordinates refer to the position of the first nucleotide 

in each recognition sequence. 


For M13mp18 – M77815; 

for M13mp19 – L08821. 


E.coli lac operon extends from nt 5869 to nt 6711. 

 

 

lac repressor binding site – 6179-6199. 

Coordinates of M13mp18/19 genes 

(termination codons included): 

I 3196-4242 

II 6849-831 

III 1579-2853 

IV 4220-5500 

V 843-1106 

VI 2856-3194 

VII 1108-1209 

VIII 1301-1522 

IX 1206-1304 

X 496-831 

Enzymes which cut M13mp18 DNA once: 

DrdI 5759 

Acc65I** 6243 

AdeI 5716 

AlfI 4843 

AloI 5765 

Alw44I* 4743 

BamHI** 6252 

BmrI 6317 

BglI 6431 

BglII 6935 

BseRI 2008 

Bsp1407I 1021 

BtsI 6088 

CaiI 2187 

Cfr9I** 6247 

Cfr10I 5613 

CspCI 3010 

Ecl136II** 6237 

Eco47I 5914 

Eco81I 6508 

Eco105I 1268 

EcoRI** 6231 

EheI 6001 

Ala Leu Ala Val Val Leu Gln LacZ 

GCA CTG GCC GTC GTT TTA CAA 


Cfr9I HincII PstI 

Ecl136II Acc65I SmaI SalI SdaI 

6231 EcoRI SacI KpnI BamHI XbaI XmiI PaeI HindIII 6287 

ACG AAT TCG AGC TCG GTA CCC GGG GAT CCT CTA GAG TCG ACC TGC AGG CAT GCA AGC TTG 

Thr Asn Ser Ser Ser Val Pro Gly Asp Pro Leu Glu Ser Thr Cys Arg His Ala Ser Leu 

Multiple Cloning Site of M13mp19 


Leu Ala Val Val Leu Gln LacZ 

CTG GCC GTC GTT TTA CAA 

CAG GAA ACA GCT ATG ACC ATG ATT ACG 

Met Thr Met Ile Thr 


PstI HincII 

SdaI SalI SmaI Acc65I Ecl136II 

6234 HindIII PaeI XmiI XbaI BamHI KpnI SacI EcoRI 6290 

CCA AGC TTG CAT GCC TGC AGG TCG ACT CTA GAG GAT CCC CGG GTA CCG AGC TCG AAT TCA 

Pro Ser Leu His Ala Cys Arg Ser Thr Leu Glu Asp Pro Arg Val Pro Ser Ser Asn Ser 

Esp3I 5971 

FalI 2486 

Hin1I 6001 

HincII** 6264 

HindIII** 6282 

KpnI** 6243 

MlsI 5080 

Mva1269I 1746 

NsbI 6425 

PacI 4132 

PaeI** 6276 

PagI 1299 

PdiI 5613 

PstI** 6270 

PvuI 6405 

SacI** 6237 

SalI** 6264 

SdaI** 6269 

SmaI** 6247 

SmiI 6783 

SspDI 6001 

XbaI** 6258 

XmiI** 

* see p.438. 

** MCS 

6264 

References 

1. Norrander, J., et 

al., Construction of 

improved M13 vectors 

using oligodeoxynucleotide-directed 

mutagenesis, Gene, 26, 

101-106, 1983. 

2. Yanisch-Perron, C., 

et al., Improved M13 

phage cloning vectors 

and host strains: 

nucleotide sequences 

of the M13mp18 and 

pUC19 vectors, Gene, 

33, 103-119, 1985. 


438 

Table 12.5. M13mp18 DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

AanI 5356 5844 

DrdI 5759 

Acc65I 6243 

AdeI 5716 

AflIII 195 3618 3717 

AlfI 4843 

AloI 5765 

Alw21I 4743 5465 6237 

Alw26I 2013 2189 4036 5972 7017 

Alw44I 4743 

BaeI 1795 2137 6517 

BamHI 6252 

BbvCI 1370 1416 

BceAI 1334 1952 3440 4719 5675 6295 6606 

BcnI 1924 6247 6248 6838 

BmrI 6317 

BfmI 205 1329 1751 2470 5495 6270 6673 

BglI 6431 

BglII 6935 

Bme1390I 1014 1924 1966 5941 5998 6137 6247 6248 6328 6455 6838 

Bpu10I 1370 1416 4012 4280 

BpuEI 868 2005 2099 2612 5370 

BsaWI 313 965 2377 2395 3369 3841 

BsaXI 1891 2667 5765 6017 

BseDI 2763 2894 5997 6136 6247 6248 6327 6617 6838 

BseGI 239 3547 6361 7244 

BseJI 1149 3974 

BseMI 5198 6913 7067 

BseRI 2008 

BseSI 2088 4743 

BseXI 932 1367 2521 3132 4871 5536 5923 6052 6355 6428 

Bsh1285I 1422 3879 6405 6521 

BshNI 1249 5677 6001 6131 6243 6465 6477 

Bsp143I 1382 1714 2221 6253 6406 6502 6936 

Bsp1407I 1021 

BspPI 1382 2221 6252 6253 

Bsu15I 2527 6882 

BtgI 2763 6617 

BtgZI 1144 1448 4971 5728 

BtsI 6088 

BveI 1113 2258 6268 

CaiI 2187 

EaeI 5080 6036 6293 

Cfr9I 6247 

Cfr10I 5613 

Cfr13I 5724 5914 5938 6396 

CseI 526 2164 2479 3237 4083 5158 6687 

CspCI 3010 

DpnI 1382 1714 2221 6253 6406 6502 6936 

DraI 189 472 4622 6784 7074 

Eam1104I 4074 6391 

EciI 1962 6603 

Ecl136II 6237 

Eco24I 5643 6237 

Eco47I 5914 

Eco47III 2710 3039 

Eco81I 6508 

Eco88I 5825 6247 

Eco105I 1268 

EcoP15I 1368 4872 4941 5894 

EcoRI 6231 

EcoRII 1014 1966 5941 5998 6137 6328 6455 

EheI 6001 

There are no restriction sites in M13mp18 DNA for the following enzymes: 

AarI, AatII, AbsI, AjiI, AjuI, ApaI, BauI, BcgI, BclI, BcuI, BfuI, BoxI, BpiI, BplI, Bpu1102I, 

BsgI, BshTI, Bsp68I, Bsp119I, Bsp120I, BspOI, BspTI, Bst1107I, BstAPI, BstXI, Cfr42I, 

CpoI, Eam1105I, Eco31I, Eco32I, Eco52I, Eco57I, Eco72I, Eco91I, Eco130I, Eco147I, 

EcoO109I, FseI, FspAI, Kpn2I, KspAI, LguI, MluI, Mph1103I, MreI, MssI, MunI, NcoI, 

NheI, NmeAIII, NotI, OliI*, PasI, PauI, Pfl23II, PfoI, Psp5II, PspXI, PsrI, PsyI, SanDI, ScaI, 

SexAI, SfaAI, SfiI, SgfI, SgrAI, SgsI, SrfI, Van91I, XagI, XcmI, XhoI, XmaJI. 



Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

Esp3I 5971 

FalI 2486 

FaqI 5090 6529 

FokI 239 3547 6361 7244 

FspBI 3827 5565 6259 6861 6979 

GsuI 6493 6899 

HaeII 2710 3039 5559 5567 6001 6446 

Hin1I 6001 

HincII 6264 

HindIII 6282 

Hpy8I 619 1022 2133 2717 4743 5713 5768 5785 5912 6264 6776 

Hpy99I 610 1950 3634 4143 6297 6310 6525 6629 

KpnI 6243 

Lsp1109I 932 1367 2521 3132 4871 5536 5923 6052 6355 6428 

LweI 25 388 1354 3979 4850 6546 6559 

MbiI 4569 5431 5572 6185 

MboI 1382 1714 2221 6253 6406 6502 6936 

MboII 781 2218 3912 4075 4271 4937 5255 5587 6391 6504 6805 

MlsI 5080 

MmeI 300 5441 5762 6627 

MspA1I 1631 5960 6053 6375 

MvaI 1014 1966 5941 5998 6137 6328 6455 

Mva1269I 1746 

NdeI 2723 3803 6846 

NmuCI 1377 1775 2543 2621 2744 5111 5541 6314 

NsbI 6425 

PacI 4132 

PaeI 6276 

PagI 1299 

PdiI 5613 

PdmI 357 2646 

Ppu21I 1268 4446 5037 5462 5716 

PscI 195 3618 3717 

Psp1406I 4634 6773 

PspFI 1192 3277 5896 

PstI 6270 

PsuI 2220 6252 6935 

PvuI 6405 

PvuII 5960 6053 6375 

RseI 2796 6996 

SacI 6237 

SalI 6264 

SchI 2011 2845 4072 5329 5766 5788 6262 6904 

SdaI 6269 

SduI 2088 4743 5465 5643 6237 

SmaI 6247 

SmiI 6783 

SmoI 868 2005 2099 2612 5370 

FauI 1385 1984 3324 5430 5517 5571 6027 6084 6398 7131 

SspI 499 2660 5023 5213 6767 6788 

SspDI 6001 

TaqI 336 1127 1508 1949 2528 3455 3694 4665 5683 6235 6265 6883 

TatI 1021 1164 1768 2132 4189 

TauI 1394 2285 2288 2312 2357 5500 5514 

TscAI 922 1786 2091 2106 4891 5350 6089 6290 

TseI 932 1367 2521 3132 4871 5536 5923 6052 6355 6428 

TspGWI 750 1159 2654 2762 2876 4352 6619 

VspI 4131 4135 4238 4628 6046 6105 6966 

XapI 1499 1674 2182 2728 3789 3900 4060 6231 6744 6755 7097 

XbaI 6258 

XceI 195 3531 3618 3717 6276 6856 

XmiI 6264 

* According to our experimental data Alw44I does not cut M13mp18/19 DNA, BseSI has only one recognition 

site in M13mp18/19 DNA, Eco47III has only one recognition site in M13mp18/19 DNA and OliI has 

one recognition site in M13mp18/19 DNA. 




pBR322 


pBR322 is one of the most commonly used E.coli cloning 

vectors. pBR322 is 4361 bp in length and contains: 

(1) the replicon rep responsible for the replication of the 

plasmid (source – plasmid pMB1); (2) the rop gene, 

coding for the Rop protein, which promotes conversion of 

the unstable RNA I – RNA II complex to a stable complex 

and serves to decrease copy number (source – plasmid 

pMB1); (3) the bla gene, coding for -lactamase, that 

confers resistance to ampicillin (source – transposon 

Tn3); (4) the tet gene, encoding tetracycline resistance 

protein (source – plasmid pSC101). 

The circular sequence is numbered such that 1 

References 

1. Bolivar, F., et al., Construction and characterization of new 

cloning vehicles. II. A multipurpose cloning system, Gene, 

2, 95-113, 1977. 

2. Covarrubias, L., et al., Construction and characterization of 

new cloning vehicles. V. Mobilization and coding properties 

of pBR322 and several deletion derivatives including 

pBR327 and pBR328, Gene, 13, 25-35, 1981. 

3. Peden, K.W., Revised sequence of the tetracyclineresistance 

gene of pBR322, Gene, 22, 277-280, 1983. 

4. Sutcliffe, J.G., Nucleotide sequence of the ampicillin 

resistance gene of Escherichia coli plasmid pBR322, Proc. 

Natl. Acad. Sci. U.S.A., 75, 3737-3741, 1978. 

5. Sutcliffe, J.G., Complete nucleotide sequence of the 

Escherichia coli plasmid pBR322, Cold Spring Harb. Symp. 

Quant. Biol., 43, Pt 1, 77-90,1979. 

6. Watson, N., A new revision of the sequence of plasmid 

pBR322, Gene, 70, 399-403, 1988. 

is the first T of the unique EcoRI site GAATTC and 

numbering increases through the tet gene, the pMB1 

material and finally through the Tn3 region. 

The plasmid map provided below lists enzymes that cut 

pBR322 DNA once. Thermo Scientific enzymes are shown 

in orange. The coordinates refer to the position of the first 


The exact positions of the genetic elements are 

shown on the map (termination codons included). 

The bla gene nucleotides 4153-4085 (complementary 

strand) code for a signal peptide. The indicated rep 

region is sufficient to promote replication. 

DNA replication initiates at position 2533 (+/- 1) and 

proceeds in the direction indicated. Plasmids carrying 

the pMB1 and ColE1 replicons are incompatible, but 

they are fully compatible with those carrying the p15A 

replicon (pACYC177, pACYC184). 

pMB1-derived plasmids can be amplified using 

chloramphenicol. 


J01749, K00005, L08654, M10282, M10283, 

M10286, M10356, M10784, M10785, M10786, 

M33694, V01119. 

Enzymes which cut pBR322 DNA once: 

AatII 4284 

AflIII 2473 

BamHI 375 

BoxI 712 

Bpu10I 1580 

BseJI 1668 

BsgI 1650 

Bsp68I 972 

BspOI 229 

Bst1107I 2244 

Bsu15I 23 

BveI 1063 

CaiI 2884 

Eam1105I 3361 

Eco31I 3433 

Eco32I 185 

Eco52I 939 

Eco88I 1425 

Eco130I 1369 

EcoRI 4359 

Esp3I 2122 

HindIII 29 

Kpn2I 1664 

LguI 2350 

MlsI 1444 

Mva1269I 1353 

NdeI 2295 

NheI 229 

PaeI 562 

PfoI 2117 

Ppu21I 2225 

PscI 2473 

PstI 3607 

PsyI 2217 

PvuI 3733 

PvuII 2064 

SalI 651 

ScaI 3844 

SgrAI 409 

SspI 4168 

VspI 3537 

XagI 622 

XapI 4359 


440 

Table 12.6. pBR322 DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

DrdI 2162 2575 

AatII 4284 

AflIII 2473 

AlfI 1026 1457 1678 

Alw21I 276 587 1174 1465 2289 2787 3948 4033 

Alw26I 2122 3433 4198 

Alw44I 2289 2787 4033 

BamHI 375 

BauI 2646 4030 4337 

BccI 458 552 988 1078 1385 1397 3402 3526 3813 

BceAI 594 1151 2959 

BcgI 708 2063 3882 

BcnI 170 534 1258 1484 1812 2118 2153 2852 3548 3899 

BmrI 304 610 679 2219 3411 

BfmI 138 2738 2929 3607 

BfuI 2682 4209 

BglI 929 1163 3480 

BoxI 712 

BpiI 737 1600 4351 

Bpu10I 1580 

BpuEI 664 870 2579 2841 3118 3986 

BsaWI 693 1664 2679 2826 3657 

BseDI 115 129 528 534 1167 1369 1447 2633 

BseGI 112 133 987 1032 1681 1770 1848 2007 2148 3346 3527 3814 

BseJI 1668 

BseMI 3420 3602 

BseMII 1581 1743 2283 2748 3157 3323 3863 

BseSI 2289 2787 4033 

BsgI 1650 

Bsh1285I 286 653 939 2386 2810 3733 3882 

BshNI 76 119 413 434 548 766 1205 1289 3314 

Bsp68I 972 

BspOI 229 

BspPI 375 376 1097 1667 3040 3114 3126 3211 3224 3688 3991 4009 

Bst1107I 2244 

BstAPI 1045 2291 

Bsu15I 23 

BtgI 528 1447 

BtgZI 205 1386 1674 

BtsI 3759 3787 

BveI 1063 

CaiI 2884 

EaeI 295 399 531 939 1444 3754 

Cfr10I 160 401 410 769 929 1283 3446 

CseI 390 649 944 976 1240 1390 2002 2179 2575 3153 3903 

Csp6I 164 2280 3845 

DraI 3230 3249 3941 

Eam1104I 2351 4155 

Eam1105I 3361 

EciI 1393 2545 2691 3519 

Eco24I 471 485 

Eco31I 3433 

Eco32I 185 

Eco47I 799 887 1136 1439 1481 1760 3504 3726 

Eco47III 232 494 775 1727 

Eco52I 939 

Eco57I 3000 4048 

Eco88I 1425 

Eco130I 1369 

EcoO109I 523 1438 1480 4341 

EcoP15I 1404 1557 1560 1686 2881 2997 3090 

EcoRI 4359 

EcoRII 130 1059 1442 2500 2621 2634 

EheI 413 434 548 1205 

Esp3I 2122 

FaqI 538 888 1084 1761 

FokI 112 133 987 1032 1681 1770 1848 2007 2148 3346 3527 3814 

There are no restriction sites in pBR322 DNA for the following enzymes: 

AanI, AarI, AbsI, Acc65I, AdeI, AjiI, AjuI, AloI, ApaI, BaeI, BbvCI, BclI, BcuI, 

BglII, BplI, Bpu1102I, BsaXI, BseRI, BshTI, Bsp119I, Bsp120I, Bsp1407I, 

BspTI, BstXI, Cfr9I, Cfr42I, CpoI, CspCI, Ecl136II, Eco105I, Eco147I, Eco72I, 

Eco81I, Eco91I, FalI, FseI, KpnI, KspAI, MauBI, MluI, Mph1103I, MreI, MssI, 

MunI, NcoI, NotI, OliI, PacI, PasI, PauI, Pfl23II, PspXI, PsrI, SacI, SanDI, SdaI, 

SexAI, SfaAI, SfiI, SgfI, SgsI, SmaI, SmiI, SrfI, XbaI, XcmI, XhoI, XmaJI. 



Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

FspAI 259 1453 

FspBI 230 1489 2968 3221 3556 

GsuI 811 1401 1981 3451 

HaeII 232 413 434 494 548 775 1205 1644 1727 2347 2717 

Hin1I 413 434 548 1205 3902 4284 

HincII 651 3905 

HindIII 29 

HinfI 632 852 1006 1304 1525 2029 2373 2448 2844 3361 

HphI 126 408 453 1307 1528 2083 2092 3217 3444 3840 4066 4081 

Hpy8I 651 1787 2244 2289 2787 3275 3905 4033 

Hpy99I 650 718 819 943 975 1241 2574 3368 3631 

HpyAV 672 690 876 993 2703 2971 3013 3484 3721 4096 

HpyF3I 1581 1743 2283 2748 3157 3323 3863 4289 

Kpn2I 1664 

LguI 2350 

MbiI 2404 4205 

MboII 464 738 1009 1601 2352 3123 3214 3969 4047 4156 4352 

MlsI 1444 

MmeI 197 284 2663 2847 

MspA1I 1139 2064 2183 2813 3058 3999 

MvaI 130 1059 1442 2500 2621 2634 

Mva1269I 1353 

NdeI 2295 

NheI 229 

NmeAIII 116 1168 3489 

NmuCI 125 213 881 1148 1915 2128 2223 3623 3834 

NsbI 260 1356 1454 3586 

PaeI 562 

PagI 489 3193 4201 4306 

PdiI 401 769 929 1283 

PdmI 2029 3961 

PfeI 852 1006 1304 1525 2029 2448 

PfoI 2117 

Ppu21I 2225 

PscI 2473 

Psp5II 1438 1480 

Psp1406I 900 1799 3591 3964 

PspFI 949 2777 

PstI 3607 

PsuI 375 1667 3114 3125 3211 3223 3991 4008 

PsyI 2217 

PvuI 3733 

PvuII 2064 

RsaI 164 2280 3845 

RseI 1027 1458 1653 2044 3614 3773 4132 

SalI 651 

ScaI 3844 

SchI 632 2373 2844 3361 

SduI 276 471 485 587 1174 1465 2289 2787 3948 4033 

SgrAI 409 

SmoI 664 870 2579 2841 3118 3986 

FauI 181 703 890 1037 1232 1497 1763 1949 2185 2195 

SspI 4168 

SspDI 413 434 548 1205 

TaiI 901 957 1546 1570 1800 2226 3176 3592 3965 4285 

TaqI 24 339 652 1127 1268 2573 4017 

TasI 59 252 1320 1334 3234 3540 3795 4360 

TatI 2279 3844 

TscAI 279 883 1132 1913 2369 2875 2888 3159 3308 3413 3760 3787 

TspDTI 86 1523 1593 1821 1861 1882 2009 3242 3344 3647 

TspGWI 1301 1610 1903 3816 4133 

Van91I 1315 1364 

VspI 3537 

XagI 622 

XapI 4359 

XceI 562 1816 2108 2473 

XmiI 651 2244 




pUC18, pUC19 


pUC18 and pUC19 vectors are small, high copy 

number, E.coli plasmids, 2686 bp in length. They are 

identical except that they contain multiple cloning sites 

(MCS) arranged in opposite orientations. pUC18/19 

plasmids contain: (1) the pMB1 replicon rep responsible 

for the replication of the plasmid (source – plasmid 

pBR322). The high copy number of pUC plasmids is 

a result of the lack of the rop gene and a single point 

mutation in the replicon rep of pMB1; (2) the bla 

gene, coding for -lactamase, that confers resistance 

to ampicillin (source – plasmid pBR322). It differs 

from that of pBR322 by two point mutations; (3) the 

region of E.coli lac operon containing a CAP protein 

binding site, promoter Plac, lac repressor binding site 

and the 5’-terminal part of the lacZ gene encoding 

the N-terminal fragment of -galactosidase (source – 

M13mp18/19). This fragment, whose synthesis 

can be induced by IPTG, is capable of intra-allelic 

Multiple Cloning Site of pUC18 

Reference 

1. Yanisch-Perron, C., et al., Improved M13 phage cloning 

vectors and host strains: nucleotide sequences of the 

M13mp18 and pUC19 vectors, Gene, 33, 103-119, 1985. 

() complementation with a defective form of 

-galactosidase encoded by the host (mutation (lacZ) 

M15). In the presence of IPTG, bacteria synthesize 

both fragments of the enzyme and form blue colonies 

on media with X-Gal. Insertion of DNA into the MCS 

located within the lacZ gene (codons 6-7 of lacZ are 

replaced by MCS) inactivates the N-terminal fragment 

of -galactosidase and abolishes -complementation. 

Bacteria carrying recombinant plasmids therefore give 

rise to white colonies. 

The map shows enzymes that cut pUC18/19 DNA 

once. Thermo Scientific enzymes are shown in orange. 

The coordinates refer to the position of the first 


The exact positions of the genetic elements are shown 

on the map (termination codons included). The bla 

gene nucleotides 2486-2418 (complementary strand) 

code for a signal peptide. The LacZ polypeptide 

corresponding to wt -galactosidase and essential for 

blue/white screening ends at nt position 236 (compl. 

strand); another 30 codons in the same reading frame 

are derived from pBR322. The indicated rep region 

is sufficient to promote replication. DNA replication 

initiates at position 866 (+/- 1) and proceeds in the 

direction indicated. Plasmids carrying the pMB1 and 

ColE1 replicons are incompatible, but they are fully 

compatible with those carrying the p15A replicon 




For pUC18 – L09136; 

for pUC19 – L09137. 


 

 

lac repressor binding site – 507-487 (compl. strand). 

Enzymes which cut pUC18 DNA once: 

AatII 2617 

Acc65I* 438 

AflIII 806 

BamHI* 429 

BcgI 2215 

BsaXI 659 

BstAPI 179 

BveI* 413 

CaiI 1217 

Cfr9I* 434 

Cfr10I 1779 

Eam1105I 1694 

Ecl136II* 444 

Eco24I* 444 

Eco31I 1766 

Eco88I* 434 

EcoO109I 2674 

EcoRI* 450 

EheI 235 

GsuI 1784 

HincII* 417 

HindIII* 399 

KpnI* 438 

LguI 683 

NdeI 183 

NmeAIII 1822 

PaeI* 405 

PdmI 2294 

PfoI 46 

PscI 806 

PspFI 1110 

PstI* 411 

SacI* 444 

SalI* 417 

ScaI 2177 

SdaI* 410 

SmaI* 434 

SspI 2501 

SspDI 235 

XapI* 450 

XbaI* 423 

XmiI* 

* MCS 

417 

M13/pUC sequencing primer, 17-mer (-20), (#S0100) PstI HincII Cfr9I Ecl136II 

SdaI SalI Eco88I Acc65I Eco24I EcoRI 

399 HindIII PaeI BveI XmiI XbaI BamHI SmaI KpnI SacI XapI 455 

5’ - G TAA AAC GAC GGC CAG TGC CAA GCT TGC ATG CCT GCA GGT CGA CTC TAG AGG ATC CCC GGG TAC CGA GCT CGA ATT CGT AAT CAT GGT CAT AGC TGT TTC CTG - 3’ 

3’ - C ATT TTG CTG CCG GTC ACG GTT CGA ACG TAC GGA CGT CCA GCT GAG ATC TCC TAG GGG CCC ATG GCT CGA GCT TAA GCA TTA GTA CCA GTA TCG ACA AAG GAC - 5’ 

Val Val Ala Leu Ala Leu Ser Ala His Arg Cys Thr Ser Glu Leu Pro Asp Gly Pro Val Ser Ser Ser Asn Thr Ile Met Thr Met 

Multiple Cloning Site of pUC19 

M13/pUC reverce sequencing primer, 17-mer (-26), (#S0101) 

M13/pUC sequencing primer, 17-mer (-20), (#S0100) Ecl136II Cfr9I HincII PstI 

EcoRI Eco24I Acc65I Eco88I SalI BveI 

396 XapI SacI KpnI SmaI BamHI XbaI XmiI SdaI PaeI HindIII 452 

5’ - G TAA AAC GAC GGC CAG TGA ATT CGA GCT CGG TAC CCG GGG ATC CTC TAG AGT CGA CCT GCA GGC ATG CAA GCT TGG CGT AAT CAT GGT CAT AGC TGT TTC CTG - 3’ 

3’ - C ATT TTG CTG CCG GTC ACT TAA GCT CGA GCC ATG GGC CCC TAG GAG ATC TCA GCT GGA CGT CCG TAC GTT CGA ACC CGA TTA GTA CCA GTA TCG ACA AAG GAC - 5’ 

Val Val Ala Leu Ala Leu Ser Ala His Arg Cys Thr Ser Glu Leu Pro Asp Gly Pro Val Ser Ser Ser Asn Thr Ile Met Thr Met 

M13/pUC reverce sequencing primer, 17-mer (-26), (#S0101) 


442 

Table 12.7. pUC18 DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

DrdI 91 908 

AatII 2617 

Acc65I 438 

AflIII 806 

Alw21I 177 444 1120 2281 2366 

Alw26I 51 1766 2531 2684 

Alw44I 177 1120 2366 

BamHI 429 

BauI 979 2363 2670 

BccI 1735 1859 2146 

BceAI 387 1292 

BcgI 2215 

BcnI 47 82 434 435 1185 1881 2232 

BmrI 364 1744 

BfmI 411 1071 1262 1940 

BfuI 1015 2542 

BglI 245 1813 

Bme1390I 47 82 354 434 435 545 833 954 967 1185 1881 2232 

BpuEI 912 1174 1451 2319 

BsaWI 1012 1159 1990 

BsaXI 659 

BseDI 354 433 434 545 966 

BseGI 77 321 1679 1860 2147 

BseLI 47 648 822 840 1006 1285 

BseMI 1753 1935 

BseMII 171 1081 1490 1656 2196 

BseNI 365 391 606 1209 1222 1339 1745 1863 1906 2170 2345 

BseSI 177 1120 2366 

BseXI 41 254 327 630 711 729 1148 1213 1216 1422 1750 2116 

Bsh1236I 2 4 107 652 654 852 1433 1763 2256 2588 

Bsh1285I 276 719 1143 2066 2215 

BshNI 235 438 550 1647 

BspLI 235 429 438 550 836 875 1647 1741 1782 1993 2583 

BspPI 429 430 1373 1447 1459 1544 1557 2021 2324 2342 

BstAPI 179 

BsuRI 287 389 646 820 831 849 1283 1741 1821 2088 2675 

BtsI 593 2092 2120 

BveI 413 

CaiI 1217 

EaeI 388 645 2087 

Cfr9I 434 

Cfr10I 1779 

Cfr13I 286 1741 1820 1837 2059 2675 

CseI 108 908 1486 2236 

Csp6I 168 439 2178 

DraI 1563 1582 2274 

Eam1104I 290 684 2488 

Eam1105I 1694 

EciI 878 1024 1852 

Ecl136II 444 

Eco24I 444 

Eco31I 1766 

Eco47I 1837 2059 

Eco57I 1333 2381 

Eco88I 434 

EcoO109I 2674 

EcoP15I 1214 1330 1423 

EcoRI 450 

EcoRII 354 545 833 954 967 

EheI 235 

Esp3I 51 2683 

FokI 77 321 1679 1860 2147 

FspBI 424 1301 1554 1889 

GsuI 1784 

HaeII 235 680 1050 

There are no restriction sites in pUC18 DNA for the following enzymes: 

AanI, AarI, AbsI, AdeI, AjiI, AjuI, AlfI, AloI, ApaI, BaeI, BbvCI, BclI, BcuI, 

BglII, BoxI, BpiI, BplI, Bpu10I, Bpu1102I, BseJI, BseRI, BsgI, BshTI, Bsp68I, 

Bsp119I, Bsp120I, Bsp1407I, BspOI, BspTI, Bst1107I, BstXI, Bsu15I, BtgI, 

BtgZI, Cfr42I, CpoI, CspCI, Eco32I, Eco47III, Eco52I, Eco72I, Eco81I, Eco91I, 

Eco105I, Eco130I, Eco147I, FalI, FaqI, FseI, FspAI, Kpn2I, KspAI, MlsI, MluI, 

Mph1103I, MreI, MssI, MunI, Mva1269I, NcoI, NheI, NotI, OliI, PacI, PasI, 

PauI, PdiI, Pfl23II, Ppu21I, Psp5II, PspXI, PsrI, PsyI, SanDI, SexAI, SfaAI, SfiI, 

SgfI, SgrAI, SgrDI, SgsI, SmiI, SrfI, Van91I, XagI, XcmI, XhoI, XmaJI. 



Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

Hin1I 235 2235 2617 

Hin1II 38 406 461 807 1527 2018 2028 2106 2142 2535 2640 

HincII 417 

HindIII 399 

HinfI 420 641 706 781 1177 1694 

HphI 12 21 1550 1777 2173 2399 2414 

Hpy8I 177 417 1120 1608 2366 

Hpy99I 372 385 907 1701 1964 

Hpy188I 31 156 918 996 1349 1483 1618 2064 2075 2195 

HpyAV 270 1036 1304 1346 1817 2054 2429 

HpyF3I 171 1081 1490 1656 2196 2622 

KpnI 438 

LguI 683 

Lsp1109I 41 254 327 630 711 729 1148 1213 1216 1422 1750 2116 

LweI 78 153 208 229 894 1946 2156 2386 

MaeIII 57 348 368 1163 1226 1342 1625 1956 2014 2167 2355 

MbiI 496 737 2538 

MboII 291 685 1456 1547 2302 2380 2489 

MmeI 996 1180 

MspA1I 112 306 628 1146 1391 2332 

MvaI 354 545 833 954 967 

NdeI 183 

NmeAIII 1822 

NmuCI 57 368 1956 2167 

NsbI 256 1919 

PaeI 405 

PagI 1526 2534 2639 

PdmI 2294 

PfeI 641 781 

PfoI 46 

PscI 806 

Psp1406I 1924 2297 

PspFI 1110 

PstI 411 

PsuI 429 1447 1458 1544 1556 2324 2341 

PvuI 276 2066 

PvuII 306 628 

RsaI 168 439 2178 

RseI 1947 2106 2465 

SacI 444 

SalI 417 

ScaI 2177 

SchI 420 706 1177 1694 

SdaI 410 

SduI 177 444 1120 2281 2366 

SmaI 434 

SmoI 912 1174 1451 2319 

FauI 114 124 284 598 655 

SspI 2501 

SspDI 235 

TaaI 19 54 262 768 839 1309 1622 2137 

TaiI 374 1509 1925 2298 2618 

TaqI 418 448 906 2350 

TasI 451 487 504 579 1567 1873 2128 

TatI 167 2177 

TauI 150 732 850 1005 2089 2211 2440 

TscAI 392 593 702 1208 1221 1492 1641 1746 2093 2120 

TseI 41 254 327 630 711 729 1148 1213 1216 1422 1750 2116 

TspDTI 639 1575 1677 1980 

TspGWI 2149 2466 

VspI 576 635 1870 

XapI 450 

XbaI 423 

XceI 37 405 806 

XmiI 417 




pUC57 


The 2710 bp pUC57 plasmid is a derivative of 

pUC19. The pUC57 MCS contains 6 restriction 

sites with protruding 3’-ends, that are resistant 

to E.coli exonuclease III. This vector is designed 

for cloning and generation of ExoIII deletions. 

The exact positions of the genetic elements 

are shown on the map (termination codons 


included). DNA replication initiates at position 

890 (+/- 1) and proceeds in the direction 

indicated. The bla gene nucleotides 2510-2442 

(compl. strand) code for a signal peptide. 


Y14837 


 

 


Enzymes which cut pUC57 DNA once: 

AatII 2641 

Acc65I* 408 

AflIII 830 

AlfI* 455 

ApaI* 442 

BamHI* 435 

BcgI 2239 

BsaXI 683 

Bsp68I* 414 

Bsp120I* 442 

BstAPI 179 

CaiI 1241 

Cfr9I* 439 

Cfr10I 1803 

Eam1105I 1718 

Ecl136II* 402 

Eco31I 1790 

Eco32I* 429 

Eco88I* 439 

Eco147I* 459 

EcoO109I 2698 

EcoRI* 396 

EheI 235 

GsuI 1808 

HincII* 448 

HindIII* 471 

KpnI* 408 

LguI 707 

Mph1103I* 420 

Mva1269I* 418 

NdeI 183 

NmeAIII 1846 

PaeI* 465 

PdmI 2318 

PfoI 46 

PscI 830 

PspFI 1134 

PstI* 453 

SacI* 402 

SalI* 448 

ScaI 2201 

SmaI* 439 

SspI 2525 

SspDI 235 

XapI* 396 

XbaI* 425 

XmiI* 448 


Eco88I ApaI 

EcoRI Ecl136II Acc65I MvaI269I Eco32I Cfr9I Bsp120I 

396 XapI SacI KpnI Bsp68I Mph1103I XbaI BamHI SmaI 

5’ - G TAA AAC GAC GGC CAG TGA ATT CGA GCT CGG TAC CTC GCG AAT GCA TCT AGA TAT CGG ATC CCG GGC CC 

3’ - C ATT TTG CTG CCG GTC ACT TAA GCT CGA GCC ATG GAG CGC TTA GCT AGA TCT ATA GCC TAG GGC CCG GG 

LacZ Val Val Ala Leu Ser Asn Ser Ser Pro Val Glu Arg Ile Cys Arg Ser Ile Pro Asp Arg Ala Arg 

HincII 

SalI AlfI 

XmiI PstI Eco174I PaeI HindIII 476 

G TCG ACT GCA GAG GCC TGC ATG CAA GCT TGG CGT AAT CAT GGT CAT AGC TGT TTC CTG - 3’ 

C AGC TGA CGT CTC CGG ACG TAC GTT CGA ACC GCA TTA GTA CCA GTA TCG ACA AAG GAC - 5’ 

Arg Ser Cys Leu Gly Ala His Leu Ser Pro Thr Ile Met Thr Met 


* MCS 


444 

Table 12.8. pUC57 DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

DrdI 91 932 

AatII 2641 

Acc65I 408 

AflIII 830 

AlfI 455 

Alw21I 177 402 1144 2305 2390 

Alw26I 51 1790 2555 2708 

Alw44I 177 1144 2390 

ApaI 442 

BamHI 435 

BauI 1003 2387 2694 

BccI 1759 1883 2170 

BceAI 387 1316 

BcgI 2239 

BcnI 47 82 439 440 1209 1905 2256 

BmrI 364 1768 

BfmI 453 1095 1286 1964 

BfuI 1039 2566 

BglI 245 1837 

Bme1390I 47 82 354 439 440 569 857 978 991 1209 1905 2256 

BpuEI 936 1198 1475 2343 

BsaWI 1036 1183 2014 

BsaXI 683 

BseDI 354 439 569 990 

BseGI 77 321 1703 1884 2171 

BseLI 47 672 846 864 1030 1309 

BseMI 1777 1959 

BseMII 171 1105 1514 1680 2220 

BseNI 365 391 630 1233 1246 1363 1769 1887 1930 2194 2369 

BseSI 177 442 1144 2390 

BseXI 41 254 327 654 735 753 1172 1237 1240 1446 1774 2140 

Bsh1236I 2 4 107 415 676 678 876 1457 1787 2280 2612 

Bsh1285I 276 743 1167 2090 2239 

BshNI 235 408 574 1671 

Bsp68I 414 

Bsp120I 442 

BspLI 235 408 435 442 574 860 899 1671 1765 1806 2017 2607 

BspPI 435 436 1397 1471 1483 1568 1581 2045 2348 2366 

BstAPI 179 

BtsI 617 2116 2144 

CaiI 1241 

EaeI 388 669 2111 

Cfr9I 439 

Cfr10I 1803 

Cfr13I 286 442 443 1765 1844 1861 2083 2699 

CseI 108 932 1510 2260 

Csp6I 168 409 2202 

DraI 1587 1606 2298 

Eam1104I 290 708 2512 

Eam1105I 1718 

EciI 902 1048 1876 

Ecl136II 402 

Eco24I 402 442 

Eco31I 1790 

Eco32I 429 

Eco47I 1861 2083 

Eco57I 1357 2405 

Eco88I 439 

Eco147I 459 

EcoO109I 2698 

EcoP15I 1238 1354 1447 

EcoRI 396 

EcoRII 354 569 857 978 991 

EheI 235 

Esp3I 51 2707 

FokI 77 321 1703 1884 2171 

FspBI 426 1325 1578 1913 

GsuI 1808 

There are no restriction sites in pUC57 DNA for the following enzymes: 

AanI, AarI, AbsI, AdeI, AjiI, AjuI, AloI, BaeI, BbvCI, BclI, BcuI, BglII, BoxI, 

BpiI, BplI, Bpu10I, Bpu1102I, BseJI, BseRI, BsgI, BshTI, Bsp119I, Bsp1407I, 

BspOI, BspTI, Bst1107I, BstXI, Bsu15I, BtgI, BtgZI, BveI, Cfr42I, CpoI, CspCI, 

Eco47III, Eco52I, Eco72I, Eco81I, Eco91I, Eco105I, Eco130I, FalI, FaqI, FseI, 

FspAI, Kpn2I, KspAI, MauBI, MlsI, MluI, MreI, MssI, MunI, NcoI, NheI, NotI, 

OliI, PacI, PasI, PauI, PdiI, Pfl23II, Ppu21I, Psp5II, PspXI, PsrI, PsyI, SanDI, 

SdaI, SexAI, SfaAI, SfiI, SgfI, SgrAI, SgrDI, SgsI, SmiI, SrfI, Van91I, XagI, 

XcmI, XhoI, XmaJI. 



Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

HaeII 235 704 1074 

Hin1I 235 2259 2641 

Hin1II 38 466 485 831 1551 2042 2052 2130 2166 2559 2664 

HincII 448 

HindIII 471 

HinfI 665 730 805 1201 1718 

HphI 12 21 1574 1801 2197 2423 2438 

Hpy8I 177 448 1144 1632 2390 

Hpy99I 372 385 447 931 1725 1988 

Hpy188I 31 156 433 942 1020 1373 1507 1642 2088 2099 2219 

HpyAV 270 1060 1370 1841 2078 2453 

HpyF3I 171 1105 1514 1680 2220 2646 

KpnI 408 

LguI 707 

Lsp1109I 41 254 327 654 735 753 1172 1237 1240 1446 1774 2140 

LweI 78 153 208 229 422 918 1970 2180 2410 

MaeIII 57 348 368 1187 1250 1366 1649 1980 2038 2191 2379 

MbiI 520 761 2562 

MboII 291 709 1328 1480 1571 2326 2404 2513 

MmeI 1020 1204 

Mph1103I 420 

MspA1I 112 306 652 1170 1415 2356 

MvaI 354 569 857 978 991 

Mva1269I 418 

NdeI 183 

NmeAIII 1846 

NmuCI 57 368 1980 2191 

NsbI 256 1943 

PaeI 465 

PagI 1550 2558 2663 

PdmI 2318 

PfeI 665 805 

PfoI 46 

PscI 830 

Psp1406I 1948 2321 

PspFI 1134 

PstI 453 

PsuI 435 1471 1482 1568 1580 2348 2365 

PvuI 276 2090 

PvuII 306 652 

RsaI 168 409 2202 

RseI 1971 2130 2489 

SacI 402 

SalI 448 

ScaI 2201 

SchI 730 1201 1718 

SduI 177 402 442 1144 2305 2390 

SmaI 439 

SmoI 936 1198 1475 2343 

FauI 114 124 284 622 679 

SspI 2525 

SspDI 235 

TaaI 19 54 262 792 863 1333 1646 2161 

TaiI 374 1533 1949 2322 2642 

TaqI 400 449 930 2374 

TasI 397 511 528 603 1591 1897 2152 

TatI 167 2201 

TauI 150 756 874 1029 2113 2235 2464 

TscAI 392 617 726 1232 1245 1516 1665 1770 2117 2144 

TseI 41 254 327 654 735 753 1172 1237 1240 1446 1774 2140 

TspDTI 663 1599 1701 2004 

TspGWI 2173 2490 

VspI 600 659 1894 

XapI 396 

XbaI 425 

XceI 37 465 830 

XmiI 448 




pTZ19R, pTZ19U 

see p.292 for pTZ19R DNA ordering information. 

pTZ19R/U are small phagemids 2862 bp in 

length, constructed by inserting the DNA of phage 

f1 intergenic region (IG) into pUC19 and the T7 

promoter sequence near to the pUC19 MCS. The 

phagemids differ in the orientation of the cloned f1 IG 

region. They are designed for DNA cloning, dideoxy 

DNA sequencing, in vitro mutagenesis and in vitro 

transcription in one system. A host strain harboring 

these phagemids will produce single-stranded DNA 

if superinfected with the helper phage M13K07. 

pTZ19R/U phagemids contain: (1) the pMB1 replicon 

rep responsible for the replication of the phagemid 

(source – plasmid pUC19); (2) the bla gene, coding 

for -lactamase, that confers resistance to ampicillin 

(source – plasmid pUC19); (3) the region of E.coli 

operon lac containing a CAP protein binding site, 

promoter Plac, lac repressor binding site and the 

5’-terminal part of lacZ gene encoding the N-terminal 

fragment of -galactosidase (source – pUC19). This 

fragment allows blue/white screening of recombinant 

phagemids in the same way as described in the 

pUC18/19 section; (4) a T7 promoter inserted near 


Reference 

1. Mead, D.A., et al., Single-stranded DNA ‘blue’ T7 promoter 

plasmids: a versatile tandem promoter system for cloning 

and protein engineering, Protein Eng., 1, 67-74, 1986. 

to the MCS of pUC19 allowing in vitro synthesis of 

large amounts of specific RNA; (5) the phage f1 

intergenic region carrying the sequences required 

in cis for initiation and termination of phage f1 DNA 

synthesis (+ and - strands) and for packaging DNA 

into bacteriophage particles. Synthesis of singlestranded 

(plus) DNA requires phage-encoded gene II, 

X and V proteins. It is initiated at ori (+) and proceeds 

in the direction indicated. The conversion of plus DNA 

strands to double-stranded DNA does not require any 

of the phage genes. DNA synthesis is initiated by a 

30-nucleotide RNA primer synthesized by the host’s 

RNA polymerase and starts at ori (-). 

The plasmid map provided below lists enzymes that cut 

pTZ19R DNA once. Thermo Scientific enzymes are shown 

in orange. The coordinates refer to the position of the first 


The exact positions of the genetic elements are 

shown on the map (termination codons included). The 

bla gene nucleotides 2732-2664 (complementary 

strand) code for a signal peptide. The LacZ 

polypeptide corresponding to wt -galactosidase 

and essential for blue/white screening ends at nt 

position 458 (complementary strand). The remaining 

amino acids in the LacZ reading frame are encoded 

by f1 DNA. The indicated rep region is sufficient to 

promote replication. DNA replication initiates in the 

complementary DNA strand at position 1112 (+/-1) 

and proceeds in the direction indicated. Phagemids 

and plasmids carrying the pMB1 and ColE1 replicons 

are incompatible with one another, but are fully 

compatible with those carrying p15A replicon 




For pTZ19R – Y14835; 

for pTZ19U – Y14836. 


 

 


 

455 (pTZ19U). 

Enzymes which cut pTZ19R DNA once: 

AanI 358 

Acc65I* 627 

AdeI 230 

AflIII 1052 

AloI 279 

BamHI* 636 

BcgI 2461 

BtgZI 242 

BveI* 652 

CaiI 1463 

Cfr9I* 631 

Eam1105I 1940 

Ecl136II* 621 

Eco31I 2012 

Eco88I* 631 

EcoRI* 615 

GsuI 2030 

Hin1I 2481 

HincII* 648 

HindIII* 666 

KpnI* 627 

LguI 929 

NmeAIII 2068 

PaeI* 660 

PdiI 127 

PdmI 2540 

Ppu21I 230 

PscI 1052 

PspFI 1356 

PstI* 654 

SacI* 621 

SalI* 648 

ScaI 2423 

SdaI* 653 

SmaI* 631 

TatI 2423 

XbaI* 642 

XmiI* 648 

M13/pUC sequencing primer, 17-mer, (-20) (#S0100) 

Cfr9I HincII PstII 

Ecl136II Acc65I Eco88I SalI BveI PaeI 

615 EcoRI SacI KpnI SmaI BamHI XbaI XmiI SdaI 

5’ - G TAA AAC GAC GGC CAG TGA ATT CGA GCT CGG TAC CCG GGG ATC CTC TAG AGT CGA CCT GCA GGC ATG C 

3’ - C ATT TTG CTG CCG GTC ACT TAA GCT CGA GCC ATG GGC CCC TAG CAG ATC TCA GCT GGA CGT CCG TAC G 

LacZ Val Val Ala Leu Ser Asn Ser Ser Pro Val Arg Pro Asp Glu Leu Thr Ser Arg Cys Ala His 

HindIII 671 

AA GCT TTC CCT ATA GTG AGT CGT ATT AGA GCT TGG CGT AAT CAT GGT CAT AGC TGT TTC CTG - 3’ 

TT CGA AAG GGA TAT CAC TCA GCA TAA TCT CGA ACC GCA TTA GTA CCA GTA TCG ACA AAG GAC - 5’ 

Leu Ser Glu Arg Tyr His Thr Thr Asn Ser Ser Pro Thr Ile Met Thr Met 

T7 transcription start 

T7 promoter 


* MCS 


446 

Table 12.9. pTZ19R DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

AanI 358 

DrdI 273 1154 

Acc65I 627 

AdeI 230 

AflIII 1052 

AloI 279 

Alw21I 621 1366 2527 2612 

Alw26I 2012 2777 

Alw44I 1366 2612 

BamHI 636 

BauI 1225 2609 

BccI 223 241 1981 2105 2392 

BceAI 189 606 1538 

BcgI 2461 

BcnI 631 632 1431 2127 2478 

BmrI 583 1990 

BfmI 9 654 675 1317 1508 2186 

BfuI 1261 2788 

BglI 464 2059 

Bme1390I 573 631 632 791 1079 1200 1213 1431 2127 2478 

BpuEI 1158 1420 1697 2565 

BsaWI 1258 1405 2236 

BsaXI 279 905 

BseDI 573 631 632 791 1212 

BseGI 540 1925 2106 2393 

BseLI 7 333 894 1068 1086 1252 1531 

BseMI 1999 2181 

BseMII 1327 1736 1902 2442 

BseNI 316 584 610 852 1455 1468 1585 1991 2109 2152 2416 2591 

BseSI 1366 2612 

BseXI 50 473 546 876 957 975 1394 1459 1462 1668 1996 2362 

Bsh1236I 3 27 47 423 898 900 1098 1679 2009 2502 2834 

Bsh1285I 495 965 1389 2312 2461 

BshNI 191 627 796 1893 

BspPI 636 637 1619 1693 1705 1790 1803 2267 2570 2588 

BsuRI 239 381 506 608 892 1066 1077 1095 1529 1987 2067 2334 

BtgZI 242 

BtsI 839 2338 2366 

BveI 652 

CaiI 1463 

EaeI 607 891 2333 

Cfr9I 631 

Cfr10I 127 2025 

Cfr13I 238 505 1987 2066 2083 2305 

CseI 1 1154 1732 2482 

Csp6I 628 2424 

DraI 1809 1828 2520 

Eam1104I 509 930 2734 

Eam1105I 1940 

EciI 1124 1270 2098 

Ecl136II 621 

Eco24I 157 621 

Eco31I 2012 

Eco47I 2083 2305 

Eco57I 1579 2627 

Eco88I 631 

EcoP15I 1460 1576 1669 

EcoRI 615 

EcoRII 573 791 1079 1200 1213 

FokI 540 1925 2106 2393 

FspBI 79 643 1547 1800 2135 

GsuI 2030 

HaeII 73 81 926 1296 

Hin1I 2481 

Hin1II 661 707 1053 1773 2264 2274 2352 2388 2781 

HincII 648 

There are no restriction sites in pTZ19R DNA for the following enzymes: 

AarI, AatII, AbsI, AjiI, AjuI, AlfI, ApaI, BaeI, BbvCI, BclI, BcuI, BglII, BoxI, BpiI, 

BplI, Bpu10I, Bpu1102I, BseJI, BseRI, BsgI, BshTI, Bsp68I, Bsp119I, Bsp120I, 

Bsp1407I, BspOI, BspTI, Bst1107I, BstAPI, BstXI, Bsu15I, BtgI, Cfr42I, CpoI, 

CspCI, Eco32I, Eco47III, Eco52I, Eco72I, Eco81I, Eco91I, Eco105I, Eco130I, 

Eco147I, EcoO109I, EheI, Esp3I, FalI, FaqI, FseI, FspAI, Kpn2I, KspAI, MauBI, 

MlsI, MluI, Mph1103I, MreI, MssI, MunI, Mva1269I, NcoI, NdeI, NheI, NotI, OliI, 

PacI, PasI, PauI, Pfl23II, PfoI, Psp5II, PspXI, PsrI, PsyI, SanDI, SexAI, SfaAI, SfiI, 

SgfI, SgrAI, SgrDI, SgsI, SmiI, SrfI, SspDI, Van91I, XagI, XcmI, XhoI, XmaJI. 



Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

HindIII 666 

HinfI 280 302 646 682 887 952 1027 1423 1940 

HpaII 128 632 770 1259 1406 1432 1622 2026 2060 2127 2237 2479 

HphI 220 1796 2023 2419 2645 2660 

Hpy8I 227 282 299 648 1366 1854 2612 

Hpy99I 591 604 1153 1947 2210 

Hpy188I 173 1164 1242 1595 1729 1864 2310 2321 2441 

Hpy188III 588 642 856 1285 1419 1517 1615 1698 1772 2031 2780 

HpyAV 106 489 1282 1592 2063 2300 2675 

HpyCH4V 548 655 663 878 1367 1663 1998 2088 2281 2369 2613 

HpyF3I 1327 1736 1902 2442 

KpnI 627 

LguI 929 

Lsp1109I 50 473 546 876 957 975 1394 1459 1462 1668 1996 2362 

LweI 1140 2192 2402 2632 

MaeIII 43 55 567 587 1409 1472 1588 1871 2202 2260 2413 2601 

MbiI 86 742 983 2784 

MboII 101 510 931 1550 1702 1793 2548 2626 2735 

MmeI 276 1242 1426 

MnlI 195 508 640 908 941 1167 1224 1491 1891 1972 2102 2308 

MspA1I 525 874 1392 1637 2578 

MspI 128 632 770 1259 1406 1432 1622 2026 2060 2127 2237 2479 

MvaI 573 791 1079 1200 1213 

NmeAIII 2068 

NmuCI 55 587 2202 2413 

NsbI 475 2165 

PaeI 660 

PagI 1772 2780 

PdiI 127 

PdmI 2540 

PfeI 887 1027 

Ppu21I 230 

PscI 1052 

Psp1406I 2170 2543 

PspFI 1356 

PstI 654 

PsuI 636 1693 1704 1790 1802 2570 2587 

PvuI 495 2312 

PvuII 525 874 

RsaI 628 2424 

RseI 2193 2352 2711 

SacI 621 

SalI 648 

ScaI 2423 

SchI 280 302 646 682 952 1423 1940 

SdaI 653 

SduI 157 621 1366 2527 2612 

SmaI 631 

SmoI 1158 1420 1697 2565 

FauI 31 85 503 844 901 

SspI 438 2747 

TaaI 256 481 1014 1085 1555 1868 2383 

TaiI 121 231 274 286 593 1755 2171 2544 

TaqI 197 619 649 1152 2596 

TasI 416 427 453 616 733 750 825 1813 2119 2374 

TatI 2423 

TauI 14 28 978 1096 1251 2335 2457 2686 

TscAI 611 839 948 1454 1467 1738 1887 1992 2339 2366 

TseI 50 473 546 876 957 975 1394 1459 1462 1668 1996 2362 

TspDTI 885 1821 1923 2226 

TspGWI 2395 2712 

VspI 822 881 2116 

XapI 415 426 615 

XbaI 642 

XceI 660 1052 

XmiI 648 




pTZ57R 

The 2886 bp pTZ57R plasmid is a derivative of 

pTZ19R. pTZ57R MCS contains 6 restriction sites 

with protruding 3’-ends, that are resistant to E.coli 

exonuclease III. This vector is designed for cloning 

and generation of ExoIII deletions. The exact position 


of genetic elements is shown on the map (termination 

codons included). DNA replication initiates at position 

1136 (+/- 1) and proceeds in the indicated direction. 

The bla gene nucleotides 2688-2756 (complementary 

strand) code for a signal peptide. 


 

 


 

Enzymes which cut pTZ57R DNA once: 

AanI 358 

Acc65I* 627 

AdeI 230 

AflIII 1076 

AlfI* 674 

AloI 279 

ApaI* 661 

BamHI* 654 

BcgI 2485 

Bsp68I* 633 

Bsp120I* 661 

BtgZI 242 

CaiI 1487 

Cfr9I* 658 

Eam1105I 1964 

Ecl136II* 621 

Eco31I 2036 

Eco32I* 648 

Eco88I* 658 

Eco147I* 678 

EcoRI* 615 

GsuI 2054 

Hin1I 2505 

HincII* 667 

HindIII* 690 

KpnI* 627 

LguI 953 

Mph1103I* 639 

Mva1269I* 637 

NmeAIII 2092 

PaeI* 684 

PdiI 127 

PdmI 2564 

Ppu21I 230 

PscI 1076 

PspFI 1380 

PstI* 672 

SacI* 621 

SalI* 667 

ScaI 2447 

SmaI* 658 

TatI 2447 

XbaI* 644 

XmiI* 667 


Eco88I ApaI 

Ecl136II Acc65I MvaI269I Eco32I Cfr9I Bsp120I 

615 EcoRI SacI KpnI Bsp68I Mph1103I XbaI BamHI SmaI 

5’ - G TAA AAC GAC GGC CAG TGA ATT CGA GCT CGG TAC CTC GCG AAT GCA TCT AGA TAT CGG ATC CCG GGC CC 

3’ - C ATT TTG CTG CCG GTC ACT TAA GCT CGA GCC ATG GAG CGC TTA CGT AGA TCT ATA GCC TAG GGC CCG GG 

LacZ Val Val Ala Leu Ser Asn Ser Ser Pro Val Glu Arg Ile Cys Arg Ser Ile Pro Asp Arg Ala Arg 

HincII 

SalI AflI 

XmiI PstI Eco174I PaeI HindIII 695 

G TCG ACT GCA GAG GCC TGC ATG CAA GCT TTC CCT ATA GTG AGT CGT ATT AGA GCT TGG CGT AAT CAT 

C AGC TGA CGT CTC CGG ACG TAC GTT CGA AAG GGA TAT CAC TCA GCA TAA TCT CGA ACC GCA TTA GTA 

Arg Ser Cys Leu Gly Ala His Leu Ser Glu Arg Tyr His Thr Thr Asn Ser Ser Pro Thr Ile Met 

T7 transcription start T7 promoter 

GGT CAT AGC TGT TTC CTG - 3’ 

CCA GTA TCG ACA AAG GAC - 5’ 

Thr Met 


* MCS 


448 

Table 12.10. pTZ57R DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

AanI 358 

DrdI 273 1178 

Acc65I 627 

AdeI 230 

AflIII 1076 

AlfI 674 

AloI 279 

Alw21I 621 1390 2551 2636 

Alw26I 2036 2801 

Alw44I 1390 2636 

ApaI 661 

BamHI 654 

BauI 1249 2633 

BccI 223 241 2005 2129 2416 

BceAI 189 606 1562 

BcgI 2485 

BcnI 658 659 1455 2151 2502 

BmrI 583 2014 

BfmI 9 672 699 1341 1532 2210 

BfuI 1285 2812 

BglI 464 2083 

Bme1390I 573 658 659 815 1103 1224 1237 1455 2151 2502 

BpuEI 1182 1444 1721 2589 

BsaWI 1282 1429 2260 

BsaXI 279 929 

BseDI 573 658 815 1236 

BseGI 540 1949 2130 2417 

BseLI 7 333 918 1092 1110 1276 1555 

BseMI 2023 2205 

BseMII 1351 1760 1926 2466 

BseNI 316 584 610 876 1479 1492 1609 2015 2133 2176 2440 2615 

BseSI 661 1390 2636 

BseXI 50 473 546 900 981 999 1418 1483 1486 1692 2020 2386 

Bsh1236I 3 27 47 423 634 922 924 1122 1703 2033 2526 2858 

Bsh1285I 495 989 1413 2336 2485 

BshNI 191 627 820 1917 

Bsp120I 661 

Bsp68I 633 

BspPI 654 655 1643 1717 1729 1814 1827 2291 2594 2612 

BtgZI 242 

BtsI 863 2362 2390 

CaiI 1487 

EaeI 607 915 2357 

Cfr9I 658 

Cfr10I 127 2049 

Cfr13I 238 505 661 662 2011 2090 2107 2329 

CseI 1 1178 1756 2506 

Csp6I 628 2448 

DraI 1833 1852 2544 

Eam1104I 509 954 2758 

Eam1105I 1964 

EciI 1148 1294 2122 

Ecl136II 621 

Eco24I 157 621 661 

Eco31I 2036 

Eco32I 648 

Eco47I 2107 2329 

Eco57I 1603 2651 

Eco88I 658 

Eco147I 678 

EcoP15I 1484 1600 1693 

EcoRI 615 

EcoRII 573 815 1103 1224 1237 

FokI 540 1949 2130 2417 

FspBI 79 645 1571 1824 2159 

GsuI 2054 

HaeII 73 81 950 1320 

Hin1I 2505 

There are no restriction sites in pTZ57R DNA for the following enzymes: 

AarI, AatII, AbsI, AjiI, AjuI, BaeI, BbvCI, BclI, BcuI, BglII, BoxI, BpiI, BplI, 

Bpu10I, Bpu1102I, BseJI, BseRI, BsgI, BshTI, Bsp119I, Bsp1407I, BspOI, 

BspTI, Bst1107I, BstAPI, BstXI, Bsu15I, BtgI, BveI, Cfr42I, CpoI, CspCI, 

Eco47III, Eco52I, Eco72I, Eco81I, Eco91I, Eco105I, Eco130I, EcoO109I, EheI, 

Esp3I, FalI, FaqI, FseI, FspAI, Kpn2I, KspAI, MauBI, MlsI, MluI, MreI, MssI, 

MunI, NcoI, NdeI, NheI, NotI, OliI, PacI, PasI, PauI, Pfl23II, PfoI, Psp5II, 

PspXI, PsrI, PsyI, SanDI, SdaI, SexAI, SfaAI, SfiI, SgfI, SgrAI, SgrDI, SgsI, SmiI, 

SrfI, SspDI, Van91I, XagI, XcmI, XhoI, XmaJI. 



Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

Hin1II 685 731 1077 1797 2288 2298 2376 2412 2805 

HincII 667 

HindIII 690 

HinfI 280 302 706 911 976 1051 1447 1964 

HpaII 128 659 794 1283 1430 1456 1646 2050 2084 2151 2261 2503 

HphI 220 1820 2047 2443 2669 2684 

Hpy8I 227 282 299 667 1390 1878 2636 

Hpy99I 591 604 666 1177 1971 2234 

Hpy188I 173 652 1188 1266 1619 1753 1888 2334 2345 2465 

Hpy188III 588 633 644 880 1309 1443 1541 1639 1722 1796 2055 2804 

HpyAV 106 489 1306 1616 2087 2324 2699 

HpyF3I 1351 1760 1926 2466 

KpnI 627 

LguI 953 

Lsp1109I 50 473 546 900 981 999 1418 1483 1486 1692 2020 2386 

LweI 641 1164 2216 2426 2656 

MaeIII 43 55 567 587 1433 1496 1612 1895 2226 2284 2437 2625 

MbiI 86 766 1007 2808 

MboII 101 510 955 1574 1726 1817 2572 2650 2759 

MmeI 276 1266 1450 

Mph1103I 639 

MspA1I 525 898 1416 1661 2602 

MspI 128 659 794 1283 1430 1456 1646 2050 2084 2151 2261 2503 

MvaI 573 815 1103 1224 1237 

Mva1269I 637 

NmeAIII 2092 

NmuCI 55 587 2226 2437 

NsbI 475 2189 

PaeI 684 

PagI 1796 2804 

PdiI 127 

PdmI 2564 

PfeI 911 1051 

Ppu21I 230 

PscI 1076 

Psp1406I 2194 2567 

PspFI 1380 

PstI 672 

PsuI 654 1717 1728 1814 1826 2594 2611 

PvuI 495 2336 

PvuII 525 898 

RsaI 628 2448 

RseI 2217 2376 2735 

SacI 621 

SalI 667 

ScaI 2447 

SchI 280 302 706 976 1447 1964 

SduI 157 621 661 1390 2551 2636 

SmaI 658 

SmoI 1182 1444 1721 2589 

FauI 31 85 503 868 925 

SspI 438 2771 

TaaI 256 481 1038 1109 1579 1892 2407 

TaiI 121 231 274 286 593 1779 2195 2568 

TaqI 197 619 668 1176 2620 

TasI 416 427 453 616 757 774 849 1837 2143 2398 

TatI 2447 

TauI 14 28 1002 1120 1275 2359 2481 2710 

TscAI 611 863 972 1478 1491 1762 1911 2016 2363 2390 

TseI 50 473 546 900 981 999 1418 1483 1486 1692 2020 2386 

TspDTI 909 1845 1947 2250 

TspGWI 2419 2736 

VspI 846 905 2140 

XapI 415 426 615 

XbaI 644 

XceI 684 1076 

XmiI 667 




pBluescript II KS(+/-), pBluescript II SK(+/-) 


All pBluescript II phagemids are 2961 bp in length. The 

structure of these phagemids is very similar to that of 

pTZ19R/U (see pTZ19R/U description for information) 

except for the MCSs and the promoters flanking the 

MCS. pBluescript II phagemids are designed for DNA 

cloning, dideoxy DNA sequencing, in vitro mutagenesis 

and in vitro transcription in a single system. The 

pBluescript II SK and KS vector series represent 

two orientations of the MCS within the lacZ gene 

encoding the N-terminal fragment of -galactosidase 

(KS represents the orientation of the MCS in which 

lacZ transcription proceeds from KpnI to SacI, while 

SK – from SacI to KpnI). (+) and (-) symbols on 

pBluescript II phagemids indicate the orientation of 

the cloned phage f1 intergenic (IG) region carrying the 

sequences required in cis for initiation and termination 

of phage f1 DNA synthesis and for packaging of DNA 

into bacteriophage particles. Synthesis of singlestranded 

DNA requires phage-encoded gene II, X and 

V proteins and is initiated at ori (+). It proceeds in 

the direction indicated. The conversion of singlestranded 

to double-stranded DNA does not require 

any of the phage genes to function. DNA synthesis is 

initiated by a 30-nucleotide RNA primer synthesized 

by the host’s RNA polymerase and starting at ori (-). 

pBluescript II phagemids contain: (1) f1 (IG) – the 

intergenic region of phage f1; (2) rep (pMB1) – the 

pMB1 replicon responsible for the replication of 

phagemid. The rep region is sufficient to promote 

replication. DNA replication initiates at position 1213 

(+/- 1) and proceeds in the direction indicated; (3) the 

bla (Ap R ) – gene, coding for -lactamase, that confers 

resistance to ampicillin. Nucleotides 2833-2765 

(complementary strand) code for a signal peptide; (4) 

lacZ – the 5’-terminal part of the lacZ gene encoding 

the N-terminal fragment of -galactosidase. This 

fragment allows blue/white screening of recombinant 

phagemids. The LacZ polypeptide corresponding to wt 

-galactosidase and essential for blue/white screening 

ends at nt position 460 (complementary strand). Other 

codons in the same reading frame come from f1 DNA. 

The plasmid maps provided below list enzymes that 

cut pBluescript II SK/KS(+) and pBluescript II SK/KS(-) 

DNA once. Thermo Scientific enzymes are shown in 

AanI 360 

Acc65I* 653 

AdeI 232 

AflIII 1153 

AloI 281 

ApaI* 659 

BamHI* 719 

BcgI 2562 

BcuI* 725 

Bsp120I* 659 

BstXI* 744 

Bsu15I* 683 

BtgI* 747 

BtgZI 244 

CaiI 1564 

Cfr9I* 713 

Cfr42I* 747 

Eam1105I 2041 

Ecl136II* 755 

Eco31I 2113 

Eco32I* 695 

Eco52I* 738 

EcoO109I* 659 

References 

1. Alting-Mees, M.A. and Short, J.M., pBluescript II: gene mapping vectors, Nucleic Acids Res., 17, 9494, 1989. 

2. Alting-Mees, et al., pBluescriptII: multifunctional cloning and mapping vectors, Meth. Enzymol., 216, 483-495, 1992. 

EcoRI* 

Hin1I 

701 

2582 

3. Short, J.M., et al., Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties, Nucleic Acids Res., 16, 7583-7600, 1988. 

orange. The coordinates refer to the position of the 

first nucleotide in each recognition sequence. The 

exact positions of the genetic elements are shown on 

the map (termination codons included). 


For pBluescript II KS(-) – X52329; 

for pBluescript II KS(+) – X52327; 

for pBluescript II SK(-) – X52330; 

for pBluescript II SK(+) – X52328. 


 

 


 

366-457. 

 

Enzymes which cut pBluescriptII KS(+) DNA once: 

AanI 97 

Acc65I* 755 

AdeI 222 

AflIII 1153 

AloI 169 

ApaI* 749 

BamHI* 689 

BcgI 2562 

BcuI* 683 

Bsp120I* 749 

BstXI* 658 

Bsu15I* 725 

BtgI* 661 

BtgZI 213 

CaiI 1564 

Cfr9I* 695 

Cfr42I* 661 

CspCI 2958 

Eam1105I 2041 

Ecl136II* 653 

Eco31I 2113 

Eco32I* 713 

Eco52I* 670 

EcoO109I* 748 

EcoRI* 707 

FaqI 457 

GsuI 2131 

Hin1I 2582 

HincII* 734 

HindIII* 719 

KpnI* 755 

LguI 1030 

NmeAIII 2169 

NotI* 669 

OliI* 659 

PdiI 328 

PdmI 2641 

Ppu21I 225 

PscI 1153 

PspXI* 739 

PstI* 701 

SacI* 653 

SalI* 734 

ScaI 2524 

SmaI* 695 

TatI 2524 

XbaI* 677 

XceI 1153 

XhoI* 740 

XmiI* 

* MCS 

734 

Enzymes which cut pBluescriptII SK(–) DNA once: 

HincII* 674 

HindIII* 689 

KpnI* 653 

LguI 1030 

NmeAIII 2169 

NotI* 737 

OliI* 745 

PdiI 129 

PdmI 2641 

Ppu21I 232 

PscI 1153 

PspXI* 667 

PspFI 1457 

PstI* 707 

SacI* 755 

SalI* 674 

ScaI 2524 

SmaI* 713 

TatI 2524 

XbaI* 731 

XceI 1153 

XhoI* 668 

XmiI* 

* MCS 

674 


450 

Multiple Cloning Site of pBluescript II KS(-), pBluescript II KS(+) 

BstXI 

BtgI 


Ecl136II Cfr42I 

T7 promoter 


615 SacI OliI 

5’ - G TAA AAC GAC GGC CAG TGA GCG CGC GTA ATA CGA CTC ACT ATA GGG CGA ATT GGA GCT CCA CCG CGG TG 

3’ - C ATT TTG CTG CCG GTC ACT CGC GCG CAT TAT GCT GAG TGA TAT CCC GCT TAA CCT CGA GGT GGC GCC AC 

LacZ Val Val Ala Leu Ser Ala Arg Tyr Tyr Ser Glu Ser Tyr Pro Ser Asn Ser Ser Trp Arg Pro Pro 

Eco52I Cfr9I 

NotI XbaI BcuI BamHI SmaI PstI EcoRI Eco32I HindIII Bsu15I 

G CGG CCG CTC TAG AAC TAG TGG ATC CCC CGG GCT GCA GGA ATT CGA TAT CAA GCT TAT CGA TAC 

C GCC GGC GAG ATC TTG ATC ACC TAG GGG GCC CGA CGT CCT TAA GCT ATA GTT CGA ATA GCT ATG 

Thr Ser Arg Ser Pro Pro Gly Pro Val Trp Ser Lys Asn Gly Lys Thr Leu Thr Leu Glu Ser Ser Pro 

T3 transcription start T3 promoter 


Multiple Cloning Site of pBluescript II SK(-), pBluescript II SK(+) 

Eco0109I 


Acc65I ApaI 

T7 promoter 

T7 transcription start 653 KpnI Bsp120I 

5’ - G TAA AAC GAC GGC CAG TGA GCG CGC GTA ATA CGA CTC ACT ATA GGG CGA ATT GGG TAC CGG GCC CCC 

3’ - C ATT TTG CTG CCG GTC ACT CGC GCG CAT TAT GCT GAG TGA TAT CCC GCT TAA CCC ATG GCC CGG GGG 

LacZ Val Val Ala Leu Ser Asn Tyr Tyr Ser Glu Ser Tyr Pro Ser Asn Pro Val Pro Gly Gly 

HincII 

PspXI SalI Cfr9I 

XhoI XmiI Bsu15I HindIII Eco32I EcoRI PstI SmaI BamHI BcuI 

CCT CGA GGT CGA CGG TAT CGA TAA GCT TGA TAT CGA ATT CCT GCA GCC CGG GGG ATC CAC TAG T 

GGA GCT CCA GCT GCC ATA GCT ATT CGA ACT ATA GCT TAA GGA CGT CGG GCC CCC TAG GTG ATC A 

Arg Ser Thr Ser Pro Ile Ser Leu Ser Ser Ile Ser Asn Arg Cys Gly Pro Pro Asp Val Leu 

BtgI 

Cfr42I 

Eco52I OliI Ecl136II 

XbaI NotI BstXI SacI 760 

TC TAG AGC GGC CGC CAC CGC GGT GGA GCT CCA GCT TTT GTT CCC TTT AGT GAG GGT TAA TTG CGC GCT 

AG ATC TCG CCG GCG GTG GCG CCA CCT CGA GGT CGA AAA CAA GGG AAA TCA CTC CCA ATT AAC GCG CGA 

Glu Leu Ala Ala Ala Val Ala Thr Ser Ser Trp Ser Lys Asn Gly Lys Thr Leu Thr Leu Glu Ser Ser 


T3 promoter 

TGG CGT AAT CAT GGT CAT AGC TGT TTC CTG - 3’ 

ACC GCA TTA GTA CCA GTA TCG ACA AAG GAC - 5’ 

Pro Thr Ile Met Thr Met 


Pro Arg Glu Leu Val Leu Pro Asp Gly Pro Ser Cys Ser Asn Ser Ile Leu Ser Ile Ser Val 

HincII Eco0109I 

SalI PspXI ApaI Acc65I 

XmiI XhoI Bsp120I KpnI 760 

CGT CGA CCT CGA GGG GGG GCC CGG TAC CCA GCT TTT GTT CCC TTT AGT GAG GGT TAA TTG CGC GCT TGG 

GCA GCT GGA GCT CCC CCC CGG GCC ATG GGT CGA AAA CAA GGG AAA TCA CTC CCA ATT AAC GCG CGA ACC 

CGT AAT CAT GGT CAT AGC TGT TTC CTG - 3’ 

GCA TTA GTA CCA GTA TCG ACA AAG GAC - 5’ 

Thr Ile Met Thr Met 

M13/pUC reverse sequencing primer, 17-mer, (-26) (#S0101)

Table 12.11. pBluescript II KS(+) DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

AanI 97 

DrdI 176 1255 

AbsI 739 

Acc65I 755 

AdeI 222 

AflIII 1153 

AloI 169 

Alw21I 653 1467 2628 2713 

Alw26I 2113 2878 

Alw44I 1467 2713 

ApaI 749 

BamHI 689 

BauI 1326 2710 

BccI 215 233 2082 2206 2493 

BceAI 267 608 1639 

BcgI 2562 

BcnI 695 696 752 1532 2228 2579 

BcuI 683 

BmrI 585 2091 

BfmI 446 638 701 1418 1609 2287 

BfuI 1362 2889 

BglI 466 2160 

Bme1390I 575 695 696 752 892 1180 1301 1314 1532 2228 2579 

BpuEI 1259 1521 1798 2666 

BsaWI 1359 1506 2337 

BsaXI 171 1006 

BseDI 575 661 694 695 892 1313 

BseGI 542 2026 2207 2494 

BseLI 117 443 739 995 1169 1187 1353 1632 

BseMI 2100 2282 

BseMII 1428 1837 2003 2543 

BseNI 140 586 612 953 1556 1569 1686 2092 2210 2253 2517 2692 

BseSI 749 1467 2713 

Bsh1285I 497 670 1066 1490 2413 2562 

BshNI 264 755 897 1994 

Bsp120I 749 

BspPI 689 690 1720 1794 1806 1891 1904 2368 2671 2689 

BstXI 658 

Bsu15I 725 

BtgI 661 

BtgZI 213 

BtsI 940 2439 2467 

CaiI 1564 

EaeI 609 670 992 2434 

Cfr9I 695 

Cfr10I 328 2126 

Cfr13I 218 507 749 750 2088 2167 2184 2406 

Cfr42I 661 

CseI 455 1255 1833 2583 

Csp6I 756 2525 

CspCI 2958 

DraI 1910 1929 2621 

Eam1104I 511 1031 2835 

Eam1105I 2041 

EciI 1225 1371 2199 

Ecl136II 653 

Eco24I 298 653 749 

Eco31I 2113 

Eco32I 713 

Eco47I 2184 2406 

Eco52I 670 

Eco57I 1680 2728 

Eco88I 695 740 

EcoO109I 748 

EcoP15I 1561 1677 1770 

EcoRI 707 

EcoRII 575 892 1180 1301 1314 

FaqI 457 

FokI 542 2026 2207 2494 

FspBI 378 678 684 1648 1901 2236 

There are no restriction sites in pBluescript II KS(+) DNA for the following enzymes: 

AarI, AatII, AjiI, AjuI, AlfI, BaeI, BbvCI, BclI, BglII, BoxI, BpiI, BplI, Bpu10I, 

Bpu1102I, BseJI, BseRI, BsgI, BshTI, Bsp68I, Bsp119I, Bsp1407I, BspTI, 

Bst1107I, BstAPI, BveI, CpoI, Eco47III, Eco72I, Eco81I, Eco91I, Eco105I, 

Eco130I, Eco147I, EheI, Esp3I, FalI, FseI, FspAI, Kpn2I, KspAI, MlsI, MluI, 

Mph1103I, MssI, MunI, Mva1269I, NcoI, NdeI, NheI, PacI, PaeI, PasI, 

Pfl23II, PfoI, Psp5II, PsrI, PsyI, SanDI, SdaI, SexAI, SfiI, SgfI, SgrAI, SgsI, 

SmiI, SrfI, SspDI, Van91I, XagI, XcmI, XmaJI. 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

GsuI 2131 

HaeII 374 382 1027 1397 

Hin1I 2582 

Hin1II 808 1154 1874 2365 2375 2453 2489 2882 

HincII 734 

HindIII 719 

HinfI 154 176 632 988 1053 1128 1524 2041 

HphI 236 1897 2124 2520 2746 2761 

Hpy8I 156 173 228 734 1467 1955 2713 

Hpy99I 593 606 733 1254 2048 2311 

Hpy188I 283 1265 1343 1696 1830 1965 2411 2422 2542 

Hpy188III 590 677 957 1386 1520 1618 1716 1799 1873 2132 2881 

HpyAV 350 491 1383 1651 1693 2164 2401 2776 

HpyCH4V 550 702 979 1468 1764 2099 2189 2382 2470 2714 

HpyF3I 1428 1837 2003 2543 

KpnI 755 

LguI 1030 

LweI 1241 2293 2503 2733 

MaeIII 401 413 569 589 1510 1573 1689 1972 2303 2361 2514 2702 

MbiI 369 673 843 1084 2885 

MboII 355 512 1032 1803 1894 2649 2727 2836 

MmeI 179 1343 1527 

MspA1I 527 661 975 1493 1738 2679 

MvaI 575 892 1180 1301 1314 

NmeAIII 2169 

NmuCI 401 589 2303 2514 

NotI 669 

NsbI 477 2266 

OliI 659 

PagI 1873 2881 

PauI 619 792 

PdiI 328 

PdmI 2641 

PfeI 988 1128 

Ppu21I 225 

PscI 1153 

Psp1406I 2271 2644 

PspXI 739 

PspFI 759 1457 

PstI 701 

PsuI 689 1794 1805 1891 1903 2671 2688 

PvuI 497 2413 

PvuII 527 975 

RsaI 756 2525 

RseI 659 2294 2453 2812 

SacI 653 

SalI 734 

ScaI 2524 

SchI 154 176 632 1053 1524 2041 

SduI 298 653 749 1467 2628 2713 

SmaI 695 

SmoI 740 1259 1521 1798 2666 

FauI 371 425 505 945 1002 

SspI 17 2848 

TaaI 200 483 731 1115 1186 1656 1969 2484 

TaiI 171 183 226 336 595 1856 2272 2645 

TaqI 260 711 726 735 741 1253 2697 

TasI 4 30 41 648 708 788 834 851 926 1914 2220 2475 

TatI 2524 

TauI 428 442 669 672 1079 1197 1352 2436 2558 2787 

TscAI 613 940 1049 1555 1568 1839 1988 2093 2440 2467 

TspDTI 986 1922 2024 2327 

TspGWI 2496 2813 

VspI 923 982 2217 

XapI 29 40 707 

XbaI 677 

XceI 1153 

XhoI 740 

XmiI 734 



substrates isolated from dam – or dcm – hosts. 


452 

Table 12.12. pBluescript II SK(-) DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

AanI 360 

DrdI 275 1255 

AbsI 667 

Acc65I 653 

AdeI 232 

AflIII 1153 

AloI 281 

Alw21I 755 1467 2628 2713 

Alw26I 2113 2878 

Alw44I 1467 2713 

ApaI 659 

BamHI 719 

BauI 1326 2710 

BccI 225 243 2082 2206 2493 

BceAI 191 608 1639 

BcgI 2562 

BcnI 657 713 714 1532 2228 2579 

BcuI 725 

BmrI 585 2091 

BfmI 11 638 707 1418 1609 2287 

BfuI 1362 2889 

BglI 466 2160 

Bme1390I 575 657 713 714 892 1180 1301 1314 1532 2228 2579 

BpuEI 1259 1521 1798 2666 

BsaWI 1359 1506 2337 

BsaXI 281 1006 

BseDI 575 713 714 747 892 1313 

BseGI 542 2026 2207 2494 

BseLI 9 335 664 995 1169 1187 1353 1632 

BseMI 2100 2282 

BseMII 1428 1837 2003 2543 

BseNI 318 586 612 953 1556 1569 1686 2092 2210 2253 2517 2692 

BseSI 659 1467 2713 

Bsh1285I 497 738 1066 1490 2413 2562 

BshNI 193 653 897 1994 

Bsp120I 659 

BspPI 719 720 1720 1794 1806 1891 1904 2368 2671 2689 

BstXI 744 

Bsu15I 683 

BtgI 747 

BtgZI 244 

BtsI 940 2439 2467 

CaiI 1564 

EaeI 609 738 992 2434 

Cfr9I 713 

Cfr10I 129 2126 

Cfr13I 240 507 659 660 2088 2167 2184 2406 

Cfr42I 747 

CseI 3 1255 1833 2583 

Csp6I 654 2525 

DraI 1910 1929 2621 

Eam1104I 511 1031 2835 

Eam1105I 2041 

EciI 1225 1371 2199 

Ecl136II 755 

Eco24I 159 659 755 

Eco31I 2113 

Eco32I 695 

Eco47I 2184 2406 

Eco52I 738 

Eco57I 1680 2728 

Eco88I 668 713 

EcoO109I 659 

EcoP15I 1561 1677 1770 

EcoRI 701 

EcoRII 575 892 1180 1301 1314 

FokI 542 2026 2207 2494 

FspBI 81 726 732 1648 1901 2236 

GsuI 758 2131 

There are no restriction sites in pBluescript II SK(-) DNA for the following enzymes: 

AarI, AatII, AjiI, AjuI, AlfI, BaeI, BbvCI, BclI, BglII, BoxI, BpiI, BplI, Bpu10I, 

Bpu1102I, BseJI, BseRI, BsgI, BshTI, Bsp68I, Bsp119I, Bsp1407I, BspTI, 

Bst1107I, BstAPI, BveI, CpoI, CspCI, Eco47III, Eco72I, Eco81I, Eco91I, 

Eco105I, Eco130I, Eco147I, EheI, Esp3I, FalI, FaqI, FseI, FspAI, Kpn2I, KspAI, 

MlsI, MluI, Mph1103I, MssI, MunI, Mva1269I, NcoI, NdeI, NheI, PacI, PaeI, 

PasI, Pfl23II, PfoI, Psp5II, PsrI, PsyI, SanDI, SdaI, SexAI, SfiI, SgfI, SgrAI, 

SgsI, SmiI, SrfI, Van91I, XagI, XcmI, XmaJI. 



Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

HaeII 75 83 1027 1397 

Hin1I 2582 

Hin1II 808 1154 1874 2365 2375 2453 2489 2882 

HincII 674 

HindIII 689 

HinfI 282 304 632 988 1053 1128 1524 2041 

HphI 222 1897 2124 2520 2746 2761 

Hpy8I 229 284 301 674 1467 1955 2713 

Hpy99I 593 606 676 1254 2048 2311 

Hpy188I 175 1265 1343 1696 1830 1965 2411 2422 2542 

Hpy188III 590 731 957 1386 1520 1618 1716 1799 1873 2132 2881 

HpyAV 108 491 1383 1651 1693 2164 2401 2776 

HpyCH4V 550 708 979 1468 1764 2099 2189 2382 2470 2714 

HpyF3I 1428 1837 2003 2543 

KpnI 653 

LguI 1030 

LweI 1241 2293 2503 2733 

MaeIII 45 57 569 589 1510 1573 1689 1972 2303 2361 2514 2702 

MbiI 88 735 843 1084 2885 

MboII 103 512 1032 1803 1894 2649 2727 2836 

MmeI 278 1343 1527 

MspA1I 527 747 975 1493 1738 2679 

MvaI 575 892 1180 1301 1314 

NmeAIII 2169 

NmuCI 57 589 2303 2514 

NotI 737 

NsbI 477 2266 

OliI 745 

PagI 1873 2881 

PauI 619 792 

PdiI 129 

PdmI 2641 

PfeI 988 1128 

Ppu21I 232 

PscI 1153 

Psp1406I 2271 2644 

PspFI 1457 

PspXI 667 

PstI 707 

PsuI 719 1794 1805 1891 1903 2671 2688 

PvuI 497 2413 

PvuII 527 975 

RsaI 654 2525 

RseI 745 2294 2453 2812 

SacI 755 

SalI 674 

ScaI 2524 

SchI 282 304 632 1053 1524 2041 

SduI 159 659 755 1467 2628 2713 

SmaI 713 

SmoI 668 1259 1521 1798 2666 

FauI 33 87 505 945 1002 

SspI 440 2848 

TaaI 258 483 678 1115 1186 1656 1969 2484 

TaiI 123 233 276 288 595 1856 2272 2645 

TaqI 199 669 675 684 699 1253 2697 

TasI 418 429 455 648 702 788 834 851 926 1914 2220 2475 

TatI 2524 

TauI 16 30 737 740 1079 1197 1352 2436 2558 2787 

TscAI 613 940 1049 1555 1568 1839 1988 2093 2440 2467 

TspDTI 986 1922 2024 2327 

TspGWI 2496 2813 

VspI 923 982 2217 

XapI 417 428 701 

XbaI 731 

XceI 1153 

XhoI 668 

XmiI 674 




pACYC177 


The 3941 bp plasmid pACYC177 is compatible 

with pMB1- or ColE1-related plasmids and can 

therefore be used with a pMB1- or ColE1derivative 

within the same cell. pACYC177 

contains: (1) the replicon rep responsible for the 

replication of plasmid (source – plasmid p15A); 

(2) the kan gene, coding for aminoglycoside 

3’-phosphotransferase, that confers resistance 

to kanamycin (source – transposon Tn903); 

(3) the bla gene, coding for -lactamase, that 

confers resistance to ampicillin (source – 

transposon Tn3). 


is the first G of the unique HincII site GTTGAC 

References 

1. Chang, A.C.Y. and Cohen, S.N., Construction and 

characterization of amplifiable multicopy DNA cloning 

vehicles derived from the P15A cryptic miniplasmid, 

J. Bacteriol., 134, 1141-1156, 1978. 

2. Mok, Y.K., et al., BsiYI, a novel thermophilic restriction 

endonuclease that recognizes 5’ CCNNNNNNNGG 3’ and 

the discovery of a wrongly sequenced site in pACYC177, 


3. Rose, R.E., The nucleotide sequence of pACYC177, 

Nucleic Acids Res., 16, 356, 1988. 

inside the bla gene and numbering increases 

through the 3’-terminal part of bla gene, the 

p15A material, the Tn903 material and finally 

through the 5’-terminal part of bla gene. The 

map shows enzymes that cut pACYC177 DNA 

once. Thermo Scientific enzymes are shown in 

orange. The coordinates refer to the position 

of the first nucleotide in each recognition 

sequence. 


are shown on the plasmid map provided below 

(termination codons included). In the bla gene, 

nucleotides 3699-3767 code for a signal 

peptide. The indicated rep region is sufficient 

to promote replication. DNA replication initiates 

at position 1330 (+/- 1) and proceeds in the 

direction indicated. 

Derivatives of p15A are normally present in 

a somewhat lower copy number than pMB1 

derivatives and cannot be amplified to the same 

extent as pMB1 derivatives. Plasmid pACYC177 

can be amplified using chloramphenicol or 

spectinomycin. 


X06402. 

Enzymes which cut pACYC177 DNA once: 

AanI 3483 

DrdI 3397 

AatII 3563 

AdeI 1818 

AjuI 2390 

AlfI 2024 

Alw44I 3814 

BamHI 3320 

BcgI 18 

BmrI 495 

BfmI 299 

BfuI 3638 

BglI 421 

BpiI 3496 

BsgI 1506 

BshNI 592 

Bsp119I 852 

Bsp68I 2009 

BspOI 1588 

Bst1107I 1577 

Bsu15I 2044 

CaiI 987 

Cfr9I 2226 

Cfr42I 1339 

Eam1105I 540 

Eco24I 2005 

Eco31I 473 

Eco57I 3799 

Eco91I 3279 

EcoO109I 3505 

Esp3I 2364 

FalI 771 

FaqI 2791 

GsuI 455 

HincII 1 

HindIII 2472 

NheI 1588 

NmeAIII 417 

NsbI 320 

PfoI 1382 

PspXI 1951 

PstI 299 

ScaI 62 

SfaAI 2350 

SgrAI 1501 

SmaI 2226 

FauI 3470 

TatI 62 

Van91I 2611 

XagI 2262 

XceI 1013 

XhoI 1952 

XmiI 1577 


454 

Table 12.13. pACYC177 DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

AanI 3483 

DrdI 3397 

AatII 3563 

AdeI 1818 

AjuI 2390 

AlfI 2024 

AluI 191 254 354 867 1100 1691 2473 2923 3045 3152 3359 3458 

Alw21I 3814 3899 

Alw26I 474 1147 1826 2365 3650 

Alw44I 3814 

BamHI 3320 

BauI 1211 1606 3006 3510 3817 

BbvCI 1594 3017 3108 

BccI 94 381 505 1650 2126 2170 2611 2963 3284 

BceAI 1330 2657 2794 

BcgI 18 

BcnI 8 359 1023 1120 2226 2227 

BmrI 495 

BfmI 299 

BfuI 3638 

BglI 421 

BpiI 3496 

Bpu10I 1594 2370 3017 3108 3211 

BpuEI 782 1033 3861 

BsaWI 249 1048 1178 1502 2489 

BsaXI 1673 2934 

BseDI 885 1224 1339 1677 1738 2225 2226 2627 2874 2935 3392 3476 

BseGI 93 380 561 1654 1979 2612 2959 3071 3113 3285 

BseLI 1221 1738 2262 2594 2611 2869 3392 3471 

BseMI 304 486 1847 

BseRI 902 969 

BseSI 3429 3814 

BseXI 124 301 490 992 995 1060 1508 

BsgI 1506 

Bsh1236I 478 802 1155 1340 1960 2010 2355 3169 3594 3926 

Bsh1285I 24 173 1064 1434 2351 3156 

BshNI 592 

BshTI 1178 1502 

Bsp68I 2009 

Bsp119I 852 

BspLI 246 457 498 592 1217 1315 1673 2939 3320 3597 

BspOI 1588 

Bst1107I 1577 

Bsu15I 2044 

BtgI 1339 1677 1738 2874 2935 3392 3476 

BtsI 119 147 2216 2295 

Cac8I 297 430 1418 1588 2007 2291 2437 3383 3469 

CaiI 987 

EaeI 152 1157 3165 3378 

Cfr9I 2226 

Cfr10I 460 945 1178 1502 2308 

Cfr13I 181 403 420 499 1343 1706 2907 3342 3506 

Cfr42I 1339 

CseI 4 754 1283 1497 3414 

Csp6I 63 2188 3251 3389 

DraI 657 676 3906 

Eam1104I 2161 3692 

Eam1105I 540 

EciI 387 1312 1393 

Eco24I 2005 

Eco31I 473 

Eco47I 181 403 1706 2907 

Eco47III 1591 3021 

Eco57I 3799 

There are no restriction sites in pACYC177 DNA for the following enzymes: 

AarI, AbsI, Acc65I, AflIII, AjiI, AloI, ApaI, BaeI, BclI, BcuI, BglII, BoxI, BplI, 

Bpu1102I, BseJI, Bsp120I, Bsp1407I, BspTI, BstAPI, BstXI, BtgZI, BveI, CpoI, 

CspCI, Ecl136II, Eco32I, Eco52I, Eco72I, Eco81I, Eco105I, Eco130I, EcoRI, 

EheI, FseI, FspAI, KpnI, Kpn2I, KspAI, LguI, MauBI, MlsI, MluI, MreI, MssI, 

MunI, NcoI, NdeI, NotI, OliI, PacI, PaeI, PasI, PauI, PdiI, Pfl23II, Ppu21I, 

PscI, Psp5II, PspFI, PsrI, PsyI, PvuII, SacI, SalI, SanDI, SdaI, SexAI, SfiI, 

SgrDI, SgsI, SmiI, SrfI, SspDI, XbaI, XcmI, XmaJI. 



Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

Eco88I 1952 2226 

Eco91I 3279 

Eco147I 1636 2976 

EcoO109I 3505 

EcoP15I 993 1492 

EcoRII 1224 1237 1372 1383 1634 2243 2600 2979 

Esp3I 2364 

FalI 771 

FaqI 2791 

FokI 93 380 561 1654 1979 2612 2959 3071 3113 3285 

FspBI 352 687 1589 3325 

GsuI 455 

HaeII 1591 3021 

Hin1I 4 3563 

HincII 1 

HindIII 2472 

Hpy8I 1 631 1182 1577 1704 2908 3394 3814 

Hpy99I 276 539 3240 3255 3405 

HpyF10VI 301 421 998 1159 1409 1638 2060 2092 2306 2969 3099 

Lsp1109I 124 301 490 992 995 1060 1508 

MbiI 1416 3471 3642 

MmeI 1027 1066 1971 2165 2571 2580 3188 

Mph1103I 2200 2466 

MspA1I 1061 1166 1339 3848 

MvaI 1224 1237 1372 1383 1634 2243 2600 2979 

Mva1269I 2238 2315 

NheI 1588 

NmeAIII 417 

NmuCI 73 284 912 2502 3280 

NsbI 320 

PagI 713 1872 3541 3646 

PdmI 1532 3882 

PfeI 1642 2265 2321 2493 2584 2971 3368 

PfoI 1382 

Psp1406I 315 3883 

PspXI 1951 

PstI 299 

PsuI 683 695 2603 3320 3839 3856 

PvuI 173 2351 

RsaI 63 2188 3251 3389 

RseI 129 288 3138 3711 

ScaI 62 

SchI 546 1031 1448 1626 2578 2987 

SduI 2005 3429 3814 3899 

SfaAI 2350 

SgrAI 1501 

SmaI 2226 

SmoI 782 1033 1952 3861 

FauI 3470 

SspI 2277 3679 

TaiI 316 732 1819 1944 3404 3564 3884 

TaqI 853 1429 1799 1953 2045 2319 2722 2815 3155 3832 

TatI 62 

TauI 29 151 1156 1220 1338 1341 1422 1958 2797 3167 3380 3741 

TseI 124 301 490 992 995 1060 1508 

TspDTI 260 563 665 1530 1874 2388 2645 2694 2710 3115 3419 

TspGWI 91 1740 2148 2185 2873 3442 3715 

Van91I 2611 

VspI 369 2552 

XagI 2262 

XapI 1967 2151 3373 3488 

XceI 1013 

XhoI 1952 

XmiI 1577 




pACYC184 


The 4245 bp plasmid, pACYC184, is compatible 

with pMB1- or ColE1-related plasmids 

and can therefore be used together with a 

pMB1- or ColE1-derivative within the same 

cell. pACYC184 contains: (1) the replicon rep 

responsible for the replication of the plasmid 

(source – plasmid p15A); (2) the tet gene, 

encoding a tetracycline resistance protein 

(source – plasmid pSC101); (3) the cat gene, 

coding for chloramphenicol acetyl transferase, 

that confers resistance to chloramphenicol 

(source – transposon Tn9). 


is the first G of the unique EcoRI site GAATTC 

References 

1. Chang, A.C.Y. and Cohen, S.N., Construction and characterization 

of amplifiable multicopy DNA cloning vehicles 

derived from the P15A cryptic miniplasmid, J. Bacteriol., 

134, 1141-1156, 1978. 

2. Rose, R.E., The nucleotide sequence of pACYC184, 

Nucleic Acids Res., 16, 355, 1988. 

inside the cat gene and numbering increases 

through the 5’-terminal part of cat gene, the 

p15A material, the tet gene and finally through 

the 3’-terminal part of cat gene. The map shows 

enzymes that cut pACYC184 DNA once. Thermo 


coordinates refer to the position of the first 



are shown on the map (termination codons 

included). The indicated rep region is sufficient 

to promote replication. DNA replication initiates 

at position 846 (+/- 1) and proceeds in the 

direction indicated. 

Derivatives of p15A are normally present in 

a somewhat lower copy number than pMB1 

derivatives and cannot be amplified to the 

same extent as pMB1 derivatives. Due to 

the presence of the cat gene on pACYC184, 

amplification of this plasmid should be 

performed using spectinomycin. 


X06403 

Enzymes which cut pACYC184 DNA once: 

DrdI 3696 

BamHI 1870 

BauI 960 

BclI 3542 

BfmI 1633 

BseJI 3442 

BseMI 4234 

BseSI 3779 

Bsp68I 2467 

Bst1107I 594 

BstAPI 2540 

Bsu15I 1518 

BtsI 3620 

BveI 2558 

Cfr42I 832 

Eam1104I 1447 

Eam1105I 3204 

Eco32I 1680 

Eco52I 2434 

Eco81I 3592 

Eco88I 2920 

EcoRI 1 

FalI 1395 

HindIII 1524 

NcoI 3945 

PaeI 2057 

PagI 1984 

PdmI 635 

PfoI 788 

PspFI 2444 

PsyI 3699 

SalI 2146 

ScaI 3831 

TatI 3831 

VspI 1406 

XagI 2117 

XbaI 1425 


456 

Table 12.14. pACYC184 DNA Restriction Sites 


Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

DrdI 3696 

AlfI 2521 2952 3452 

Alw21I 490 1771 2082 2669 2960 

Alw26I 325 1025 4028 

BamHI 1870 

BauI 960 

BccI 475 1953 2047 2483 2573 2880 2892 3872 

BceAI 92 556 842 2089 2646 3881 

BcgI 2203 3297 

BclI 3542 

BcnI 266 358 1052 1149 1665 2029 2753 2979 3379 3701 

BmrI 1799 2105 2174 

BfmI 1633 

BglI 2424 2658 

BoxI 2207 3301 

BpiI 2232 3095 3326 

Bpu10I 228 3075 3578 

BpuEI 1138 1389 2159 2365 

BsaWI 5 669 993 1123 2188 3182 3269 3282 3438 3627 3642 

BseGI 8 1607 1628 2482 2527 3455 3734 

BseMI 4234 

BseMII 1045 1308 3076 3206 3243 3579 3593 4026 

BseRI 1202 1269 

BseSI 3779 

BsgI 665 3424 

Bsh1285I 737 1107 1781 2148 2434 3698 

BshNI 1571 1614 1908 1929 2043 2261 2700 2784 3355 3780 

BshTI 669 993 3182 3642 

Bsp68I 2467 

Bsp119I 1319 3717 

BspOI 583 1724 

BspPI 1870 1871 2592 3441 

Bst1107I 594 

BstAPI 2540 

Bsu15I 1518 

BtgI 832 2023 2942 3384 3566 3945 

BtgZI 1700 2881 3448 3825 4152 

BtsI 3620 

BveI 2558 

CaiI 532 1181 3519 

EaeI 1014 1790 1894 2026 2434 2939 3381 3981 

Cfr10I 669 993 1226 1655 1896 1905 2264 2424 2778 3182 3358 3642 

Cfr13I 346 829 1667 2019 2294 2382 2631 2755 2934 2976 3389 

Cfr42I 832 

CseI 437 675 889 1885 2144 2439 2471 2735 2885 

Csp6I 125 1659 3832 

DraI 83 3989 

Eam1104I 1447 

Eam1105I 3204 

EciI 778 859 2888 

Eco24I 1966 1980 

Eco32I 1680 

Eco47I 2294 2382 2631 2934 2976 

Eco47III 580 1727 1989 2270 3501 

Eco52I 2434 

Eco57I 1464 3151 

Eco81I 3592 

Eco88I 2920 

Eco130I 2864 3945 

EcoO109I 2018 2933 2975 3388 

EcoP15I 679 1178 2899 3052 3055 3148 3460 

EcoRI 1 

EcoRII 349 377 789 800 935 948 1625 2554 2937 3767 4015 4071 

EheI 1908 1929 2043 2700 

Esp3I 324 4028 

FalI 1395 

There are no restriction sites in pACYC184 DNA for the following enzymes: 

AanI, AarI, AatII, AbsI, Acc65I, AdeI, AflIII, AjiI, AjuI, AloI, Alw44I, ApaI, BaeI, 

BbvCI, BcuI, BfuI, BglII, BplI, Bpu1102I, BsaXI, Bsp120I, Bsp1407I, BspTI, 

BstXI, Cfr9I, CpoI, CspCI, Ecl136II, Eco31I, Eco72I, Eco91I, Eco105I, Eco147I, 

FseI, KpnI, KspAI, LguI, MauBI, MluI, Mph1103I, MssI, MunI, NdeI, NotI, 

OliI, PacI, PauI, Pfl23II, PscI, PspXI, PsrI, PstI, PvuI, SacI, SanDI, SdaI, SexAI, 

SfaAI, SfiI, SgfI, SgrDI, SgsI, SmaI, SmiI, SrfI, XcmI, XhoI, XmaJI. 



Enzyme 

1 2 3 4 5 6 7 8 9 10 11 12 

FaqI 371 520 2033 2383 2579 3136 3375 

FokI 8 1607 1628 2482 2527 3455 3734 

FspAI 1754 2948 

FspBI 584 1426 1725 2984 

GsuI 493 2306 2896 4145 

HaeII 580 1727 1908 1929 1989 2043 2270 2700 3364 3418 3501 

Hin1I 1908 1929 2043 2700 

HincII 2146 3212 

HindIII 1524 

HinfI 724 1141 2127 2347 2501 2799 3020 3210 3897 

Hpy8I 594 989 2146 3212 3646 4198 

Hpy99I 438 2145 2213 2314 2438 2470 2736 3307 3695 4131 

Hpy188I 886 1105 1309 1355 1411 1692 1768 1779 2344 3242 

HpyAV 1317 2167 2185 2371 2488 3279 

HpyF3I 229 1045 1308 3076 3206 3243 3579 3593 4026 

Kpn2I 5 3438 

MbiI 755 3223 

MboII 404 793 804 1394 1448 1959 2233 2504 3096 3327 3964 

MlsI 2939 3981 

MmeI 298 1105 1144 1692 1779 

MseI 84 812 1231 1407 1529 1551 3494 3675 3775 3796 3848 3990 

MspA1I 103 515 832 1005 1110 2634 3263 3904 

MvaI 349 377 789 800 935 948 1625 2554 2937 3767 4015 4071 

Mva1269I 14 2848 3852 

NcoI 3945 

NheI 583 1724 

NmeAIII 1611 2663 

NmuCI 410 1260 1620 1708 2376 2643 3616 

NsbI 1755 2851 2949 

PaeI 2057 

PagI 1984 

PasI 348 4014 

PdiI 1896 2264 2424 2778 3358 

PdmI 635 

PfeI 2347 2501 2799 3020 3897 

PfoI 788 

Ppu21I 310 3142 

Psp5II 2933 2975 

Psp1406I 2395 4160 

PspFI 2444 

PsuI 1870 3441 

PsyI 3699 

PvuII 103 515 

RsaI 125 1659 3832 

RseI 285 2522 2953 3427 

SalI 2146 

ScaI 3831 

SchI 724 1141 2127 3210 

SduI 490 1771 1966 1980 2082 2669 2960 3779 

SgrAI 668 1904 

SmoI 1138 1389 2159 2365 

FauI 25 1676 2198 2385 2532 2727 2992 3160 3292 3811 

SspI 1419 3936 

SspDI 1908 1929 2043 2700 

TaiI 311 323 2396 2452 3041 3065 3143 3986 4161 

TaqI 246 744 1320 1519 1834 2147 2622 2763 3706 3718 

TatI 3831 

Tru1I 84 812 1231 1407 1529 1551 3494 3675 3775 3796 3848 3990 

TspDTI 17 283 642 1581 3018 3088 3732 3844 3889 4155 4232 

TspGWI 407 2796 3105 4243 

Van91I 337 2810 2859 4015 

VspI 1406 

XagI 2117 

XapI 1 3708 3720 

XbaI 1425 

XceI 1158 2057 

XmiI 594 2146 




Number of Recognition Sites in DNA Molecules 

Table 12.15. Number of recognition sites in DNA molecules. 

Enzyme Specificity 

pJET1.2 

Lambda DNA 

X174 

M13mp18 

M13mp19 

pBR322 

pUC18 

pUC19 

pUC57 

pTZ19R 

pTZ19U 

pTZ57R 

pBluescript II 

KS(-) 


KS(+) 


SK(-) 


SK(+) 

AanI TTATAA 0 12 1 2 2 0 0 0 0 1 1 1 1 1 1 1 1 0 

AarI CACCTGC(4/8) 0 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

DrdI GACNNNNNNGTC 1 3 1 1 1 2 2 2 2 2 2 2 2 2 2 2 1 1 

AatII GACGTC 2 10 1 0 0 1 1 1 1 0 0 0 0 0 0 0 1 0 

AbsI CCTCGAGG 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 

Acc65I GGTACC 0 2 0 1 1 0 1 1 1 1 1 1 1 1 1 1 0 0 

AdeI CACNNNGTG 1 10 1 1 1 0 0 0 0 1 1 1 1 1 1 1 1 0 

AflIII ACRYGT 2 20 2 3 3 1 1 1 1 1 1 1 1 1 1 1 0 0 

AjiI CACGTC(-3/-3) 0 17 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

AjuI (7/12)GAA(N)7TTGG(11/6) 0 3 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 

AlfI (10/12)GCA(N)6TGC(12/10) 0 22 0 1 1 3 0 0 1 0 0 1 0 0 0 0 1 3 

AloI (7/12)GAAC(N) 6TCC(12/7) 1 7 0 1 1 0 0 0 0 1 1 1 1 1 1 1 0 0 

AluI AGCT 14 143 24 27 27 17 16 16 16 17 17 17 17 17 17 17 12 13 

Alw21I GWGCWC 3 28 3 3 3 8 5 5 5 4 4 4 4 4 4 4 2 5 

Alw26I GTCTC(1/5) 4 37 4 5 5 3 4 4 4 2 2 2 2 2 2 2 5 3 

Alw44I GTGCAC 2 4 1 1* 1* 3 3 3 3 2 2 2 2 2 2 2 1 0 

ApaI GGGCCC 0 1 0 0 0 0 0 0 1 0 0 1 1 1 1 1 0 0 

BaeI (10/15)AC(N)4GTAYC(12/7) 0 10 1 3 3 0 0 0 0 0 0 0 0 0 0 0 0 0 

BamHI GGATCC 0 5 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

BauI CACGAG(-5/-1) 3 8 2 0 0 3 3 3 3 2 2 2 2 2 2 2 5 1 

BbvCI CCTCAGC(-5/-2) 1 7 3 2 2 0 0 0 0 0 0 0 0 0 0 0 3 0 

BccI CCATC(4/5) 5 145 12 14 14 9 3 3 3 5 5 5 5 5 5 5 9 8 

BceAI ACGGC(12/14) 1 115 11 7 7 3 2 2 2 3 3 3 3 3 3 3 3 6 

BcgI (10/12)CGA(N)6TGC(12/10) 1 28 2 0 0 3 1 1 1 1 1 1 1 1 1 1 1 2 

BclI TGATCA 0 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 

BcnI CCSGG 3 114 1 4 4 10 7 7 7 5 5 5 6 6 6 6 6 10 

BcuI ACTAGT 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 

BmrI ACTGGG(5/4) 1 4 0 1 1 5 2 2 2 2 2 2 2 2 2 2 1 3 

BfmI CTRYAG 5 38 6 7 7 4 4 4 4 6 6 6 6 6 6 6 1 1 

BfuI GTATCC(6/5) 2 26 4 0 0 2 2 2 2 2 2 2 2 2 2 2 1 0 

BglI GCCNNNNNGGC 1 29 0 1 1 3 2 2 2 2 2 2 2 2 2 2 1 2 

BglII AGATCT 2 6 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 

Bme1390I CCNGG 7 185 3 11 11 16 12 12 12 10 10 10 11 11 11 11 14 22 

BoxI GACNNNNGTC 0 7 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 

BpiI GAAGAC(2/6) 1 24 3 0 0 3 0 0 0 0 0 0 0 0 0 0 1 3 

BplI (8/13)GAG(N) 5CTC(13/8) 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Bpu10I CCTNAGC(-5/-2) 1 19 7 4 4 1 0 0 0 0 0 0 0 0 0 0 5 3 

Bpu1102I GCTNAGC 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

BpuEI CTTGAG(16/14) 5 13 2 5 5 6 4 4 4 4 4 4 4 4 4 4 3 4 

BsaWI WCCGGW 4 81 3 6 6 5 3 3 3 3 3 3 3 3 3 3 5 11 

BsaXI (9/12)AC(N) 5CTCC(10/7) 1 19 0 4 4 0 1 1 1 2 2 2 2 2 2 2 2 0 

BseDI CCNNGG 3 105 6 9 9 8 5 5 4 5 5 4 6 6 6 6 12 18 

BseGI GGATG(2/0) 6 150 8 4 4 12 5 5 5 4 4 4 4 4 4 4 10 7 

BseJI GATNNNNATC 0 21 2 2 2 1 0 0 0 0 0 0 0 0 0 0 0 1 

BseLI CCNNNNNNNGG 5 176 19 17 17 20 6 6 6 7 7 7 8 8 8 8 8 21 

BseMI GCAATG(2/0) 2 44 4 3 3 2 2 2 2 2 2 2 2 2 2 2 3 1 

BseMII CTCAG(10/8) 7 80 10 23 23 7 5 5 5 4 4 4 4 4 4 4 13 8 

BseNI ACTGG(1/-1) 9 110 9 18 18 19 11 11 11 12 12 12 12 12 12 12 14 15 

BseRI GAGGAG(10/8) 0 19 2 1 1 0 0 0 0 0 0 0 0 0 0 0 2 2 

BseSI GKGCMC 2 10 1 2* 2* 3 3 3 4 2 2 3 3 3 3 3 2 1 

BseXI GCAGC(8/12) 11 199 14 10 10 21 12 12 12 12 12 12 13 13 13 13 7 15 

BsgI GTGCAG(16/14) 0 41 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 2 

Bsh1236I CGCG 7 157 14 18 18 23 10 10 11 11 11 12 15 15 15 15 10 18 

Bsh1285I CGRYCG 5 22 1 4 4 7 5 5 5 5 5 5 6 6 6 6 6 6 

BshNI GGYRCC 2 25 3 7 7 9 4 4 4 4 4 4 4 4 4 4 1 10 

BshTI ACCGGT 0 13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 4 

Bsp119I TTCGAA 0 7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2 

Bsp120I GGGCCC 0 1 0 0 0 0 0 0 1 0 0 1 1 1 1 1 0 0 

Bsp1407I TGTACA 0 5 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 

Bsp143I GATC 15 116 0 7 7 22 15 15 15 15 15 15 15 15 15 15 22 15 


pACYC177 

pACYC184 


458 




pJET1.2 

Lambda DNA 

X174 

M13mp18 

M13mp19 

pBR322 

pUC18 

pUC19 

pUC57 

pTZ19R 

pTZ19U 

pTZ57R 


KS(-) 


KS(+) 


SK(-) 


SK(+) 

Bsp68I TCGCGA 0 5 2 0 0 1 0 0 1 0 0 1 0 0 0 0 1 1 

BspLI GGNNCC 10 82 6 18 18 24 11 11 12 13 13 14 15 15 15 15 10 23 

BspOI GCTAGC 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 2 

BspPI GGATC(4/5) 8 58 0 4 4 12 10 10 10 10 10 10 10 10 10 10 13 4 

BspTI CTTAAG 0 3 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Bst1107I GTATAC 0 3 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 1 

BstAPI GCANNNNNTGC 0 34 2 0 0 2 1 1 1 0 0 0 0 0 0 0 0 1 

BstXI CCANNNNNNTGG 0 13 3 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 

Bsu15I ATCGAT 1 15 0 2 2 1 0 0 0 0 0 0 1 1 1 1 1 1 

BsuRI GGCC 10 149 11 15 15 22 11 11 13 12 12 14 14 14 14 14 13 24 

BtgI CCRYGG 1 46 3 2 2 2 0 0 0 0 0 0 1 1 1 1 7 6 

BtgZI GCGATG(10/14) 0 45 2 4 4 3 0 0 0 1 1 1 1 1 1 1 0 5 

BtsI GCAGTG(2/0) 3 34 1 1 1 2 3 3 3 3 3 3 3 3 3 3 4 1 

BveI ACCTGC(4/8) 1 41 3 3 3 1 1 1 0 1 1 0 0 0 0 0 0 1 

Cac8I GCNNGC 7 238 19 28 28 31 14 14 14 16 16 16 15 15 15 15 9 30 

CaiI CAGNNNCTG 1 41 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 

EaeI YGGCCR 3 39 2 3 3 6 3 3 3 3 3 3 4 4 4 4 4 8 

Cfr9I CCCGGG 0 3 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 

Cfr10I RCCGGY 1 61 0 1 1 7 1 1 1 2 2 2 2 2 2 2 5 12 

Cfr13I GGNCC 4 74 2 4 4 15 6 6 8 6 6 8 8 8 8 8 9 11 

Cfr42I CCGCGG 0 4 1 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 

CpoI CGGWCCG 0 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

CseI GACGC(5/10) 3 102 14 7 7 11 4 4 4 4 4 4 4 4 4 4 5 9 

Csp6I GTAC 1 113 11 19 19 3 3 3 3 2 2 2 2 2 2 2 4 3 

CspCI (11/13)CAA(N)5GTGG(12/10) 0 7 2 1 1 0 0 0 0 0 1 0 0 1 0 1 0 0 

CviJI RGCY 41 692 69 102 102 73 45 45 47 46 46 48 49 49 49 49 54 72 

DpnI GATC 15 116 0 7 7 22 15 15 15 15 15 15 15 15 15 15 22 15 

DraI TTTAAA 7 13 2 5 5 3 3 3 3 3 3 3 3 3 3 3 3 2 

Eam1104I CTCTTC(1/4) 2 34 2 2 2 2 3 3 3 3 3 3 3 3 3 3 2 1 

Eam1105I GACNNNNNGTC 1 9 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 

EciI GGCGGA(11/9) 3 32 2 2 2 4 3 3 3 3 3 3 3 3 3 3 3 3 

Ecl136II GAGCTC 0 2 0 1 1 0 1 1 1 1 1 1 1 1 1 1 0 0 

Eco24I GRGCYC 0 7 0 2 2 2 1 1 2 2 2 3 3 3 3 3 1 2 

Eco31I GGTCTC(1/5) 1 2 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 

Eco32I GATATC 1 21 0 0 0 1 0 0 1 0 0 1 1 1 1 1 0 1 

Eco47I GGWCC 2 35 1 1 1 8 2 2 2 2 2 2 2 2 2 2 4 5 

Eco47III AGCGCT 0 2 0 2* 2* 4 0 0 0 0 0 0 0 0 0 0 2 5 

Eco52I CGGCCG 1 2 0 0 0 1 0 0 0 0 0 0 1 1 1 1 0 1 

Eco57I CTGAAG(16/14) 4 40 0 0 0 2 2 2 2 2 2 2 2 2 2 2 1 2 

Eco72I CACGTG 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

Eco81I CCTNAGG 0 2 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 

Eco88I CYCGRG 1 8 1 2 2 1 1 1 1 1 1 1 2 2 2 2 2 1 

Eco91I GGTNACC 0 13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 

Eco105I TACGTA 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 

Eco130I CCWWGG 1 10 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 

Eco147I AGGCCT 0 6 1 0 0 0 0 0 1 0 0 1 0 0 0 0 2 0 

EcoO109I RGGNCCY 0 3 0 0 0 4 1 1 1 0 0 0 1 1 1 1 1 4 

EcoP15I CAGCAG(25/27) 3 72 5 4 4 7 3 3 3 3 3 3 3 3 3 3 2 7 

EcoRI GAATTC 0 5 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 

EcoRII CCWGG 4 71 2 7 7 6 5 5 5 5 5 5 5 5 5 5 8 12 

EheI GGCGCC 0 1 2 1 1 4 1 1 1 0 0 0 0 0 0 0 0 4 

Esp3I CGTCTC(1/5) 1 14 0 1 1 1 2 2 2 0 0 0 0 0 0 0 1 2 

FalI (8/13)AAG(N)5CTT(13/8) 0 15 1 1 1 0 0 0 0 0 0 0 0 0 0 0 1 1 

FaqI GGGAC(10/14) 1 38 2 2 2 4 0 0 0 0 1 0 0 1 0 1 1 7 

FokI GGATG(9/13) 6 150 8 4 4 12 5 5 5 4 4 4 4 4 4 4 10 7 

FseI GGCCGGCC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

FspAI RTGCGCAY 0 2 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 2 

FspBI CTAG 4 13 3 5 5 5 4 4 4 5 5 5 6 6 6 6 4 4 

GsuI CTGGAG(16/14) 1 25 3 2 2 4 1 1 1 1 1 1 1 1 2 2 1 4 

HaeII RGCGCY 2 48 8 6 6 11 3 3 3 4 4 4 4 4 4 4 2 11 

HhaI GCGC 13 215 18 26 26 31 17 17 17 20 20 20 24 24 24 24 16 26 

Hin1I GRCGYC 3 40 7 1 1 6 3 3 3 1 1 1 1 1 1 1 2 4 


pACYC177 

pACYC184



pJET1.2 

Lambda DNA 

X174 

M13mp18 

M13mp19 

pBR322 

pUC18 

pUC19 

pUC57 

pTZ19R 

pTZ19U 

pTZ57R 


KS(-) 


KS(+) 


SK(-) 


SK(+) 

Hin1II CATG 11 181 22 15 15 26 11 11 11 9 9 9 8 8 8 8 16 23 

Hin6I GCGC 13 215 18 26 26 31 17 17 17 20 20 20 24 24 24 24 16 26 

HincII GTYRAC 0 35 13 1 1 2 1 1 1 1 1 1 1 1 1 1 1 2 

HindIII AAGCTT 1 6 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

HinfI GANTC 7 148 21 27 27 10 6 6 5 9 9 8 8 8 8 8 13 9 

HpaII CCGG 12 328 5 18 18 26 13 13 13 12 12 12 13 13 13 13 16 34 

HphI GGTGA(8/7) 5 168 9 18 18 12 7 7 7 6 6 6 6 6 6 6 17 16 

Hpy8I GTNNAC 3 125 23 11 11 8 5 5 5 7 7 7 7 7 7 7 8 6 

Hpy99I CGWCG 3 102 8 8 8 9 5 5 6 5 5 6 6 6 6 6 5 10 

Hpy188I TCNGA 9 170 18 31 31 15 10 10 11 9 9 10 9 9 9 9 18 10 

Hpy188III TCNNGA 19 185 26 28 28 19 13 13 14 11 11 12 11 11 11 11 22 14 

HpyAV CCTTC(6/5) 6 106 14 14 14 10 7 6 6 7 7 7 8 8 8 8 13 6 

HpyCH4V TGCA 11 273 18 18 18 21 13 13 15 11 11 13 10 10 10 10 17 17 

HpyF3I CTNAG 9 104 14 29 29 8 6 6 6 4 4 4 4 4 4 4 14 9 

HpyF10VI GCNNNNNNNGC 9 347 21 20 20 34 13 13 13 14 14 14 16 16 16 16 11 36 

KpnI GGTACC 0 2 0 1 1 0 1 1 1 1 1 1 1 1 1 1 0 0 

Kpn2I TCCGGA 1 24 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 2 

KspAI GTTAAC 0 14 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

LguI GCTCTTC(1/4) 1 10 1 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 

Lsp1109I GCAGC(8/12) 11 199 14 10 10 21 12 12 12 12 12 12 13 13 13 13 7 15 

LweI GCATC(5/9) 6 169 12 7 7 22 8 8 9 4 4 5 4 4 4 4 17 16 

MaeIII GTNAC 8 156 17 24 24 17 11 11 11 12 12 12 12 12 12 12 14 13 

MauBI CGCGCGCG 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

MbiI CCGCTC(-3/-3) 4 17 1 4 4 2 3 3 3 4 4 4 5 5 5 5 3 2 

MboI GATC 15 116 0 7 7 22 15 15 15 15 15 15 15 15 15 15 22 15 

MboII GAAGA(8/7) 12 130 11 11 11 11 7 8 8 9 9 9 8 8 8 8 15 11 

MlsI TGGCCA 0 18 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 2 

MluI ACGCGT 0 7 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

MmeI TCCRAC(20/18) 2 18 5 4 4 4 2 2 2 3 3 3 3 3 3 3 7 5 

MnlI CCTC(7/6) 15 262 34 61 61 26 13 13 14 12 12 13 14 14 14 14 26 27 

Mph1103I ATGCAT 0 14 0 0 0 0 0 0 1 0 0 1 0 0 0 0 2 0 

MreI CGCCGGCG 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

MseI TTAA 23 195 35 63 63 15 13 13 13 18 18 18 19 19 19 19 16 12 

MspA1I CMGCKG 5 75 5 4 4 6 6 6 6 5 5 5 6 6 6 6 4 8 

MspI CCGG 12 328 5 18 18 26 13 13 13 12 12 12 13 13 13 13 16 34 

MssI GTTTAAAC 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

MunI CAATTG 1 8 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

MvaI CCWGG 4 71 2 7 7 6 5 5 5 5 5 5 5 5 5 5 8 12 

Mva1269I GAATGC(1/-1) 1 46 4 1 1 1 0 0 1 0 0 1 0 0 0 0 2 3 

NcoI CCATGG 1 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 

NdeI CATATG 0 7 0 3 3 1 1 1 1 0 0 0 0 0 0 0 0 0 

NheI GCTAGC 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 2 

NmeAIII GCCGAG(21/19) 1 8 3 0 0 3 1 1 1 1 1 1 1 1 1 1 1 2 

NmuCI GTSAC 2 81 8 8 8 9 4 4 4 4 4 4 4 4 4 4 5 7 

NotI GCGGCCGC 1 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 

NsbI TGCGCA 1 15 1 1 1 4 2 2 2 2 2 2 2 2 2 2 1 3 

OliI CACNNNNGTG 0 20 1 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 

PacI TTAATTAA 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 

PaeI GCATGC 0 6 0 1 1 1 1 1 1 1 1 1 0 0 0 0 0 1 

PagI TCATGA 4 8 3 1 1 4 3 3 3 2 2 2 2 2 2 2 4 1 

PasI CCCWGGG 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 

PauI GCGCGC 0 6 1 0 0 0 0 0 0 0 0 0 2 2 2 2 0 0 

PdiI GCCGGC 0 1 0 1 1 4 0 0 0 1 1 1 1 1 1 1 0 5 

PdmI GAANNNNTTC 1 24 3 2 2 2 1 1 1 1 1 1 1 1 1 1 2 1 

PfeI GAWTC 2 87 11 19 19 6 2 2 2 2 2 2 2 2 2 2 7 5 

Pfl23II CGTACG 0 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

PfoI TCCNGGA 0 15 0 0 0 1 1 1 1 0 0 0 0 0 0 0 1 1 

Ppu21I YACGTR 0 14 2 5 5 1 0 0 0 1 1 1 1 1 1 1 0 2 

PscI ACATGT 2 2 0 3 3 1 1 1 1 1 1 1 1 1 1 1 0 0 

Psp5II RGGWCCY 0 3 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 2 

Psp1406I AACGTT 2 7 3 2 2 4 2 2 2 2 2 2 2 2 2 2 2 2 

PspFI CCCAGC(-1/-5) 1 32 2 3 3 2 1 1 1 1 1 1 2 2 1 1 0 1 


pACYC177 

pACYC184 


460 




R = G or A; H = A, C or T; N = G, A, T or C. 

Y = C or T; V = A, C or G; K = G or T; 

W = A or T; B = C, G or T; D = A, G or T; 

M = A or C. 


pJET1.2 

Lambda DNA 

X174 

M13mp18 

M13mp19 

pBR322 

pUC18 

pUC19 

pUC57 

* According to our experimental data: 

Alw44I does not cut M13mp18/19 DNA. 

BseSI has only one recognition site in M13mp18/19 DNA. 

Eco47III has only one recognition site in M13mp18/19 DNA. 

pTZ19R 

pTZ19U 

pTZ57R 


KS(-) 


KS(+) 


SK(-) 


SK(+) 

PspXI VCTCGAGB 1 1 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 0 

PsrI (7/12)GAAC(N)6TAC(12/7) 0 3 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

PstI CTGCAG 1 28 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 

PsuI RGATCY 8 21 0 3 3 8 7 7 7 7 7 7 7 7 7 7 6 2 

PsyI GACNNNGTC 0 2 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 

PvuI CGATCG 1 3 0 1 1 1 2 2 2 2 2 2 2 2 2 2 2 0 

PvuII CAGCTG 2 15 0 3 3 1 2 2 2 2 2 2 2 2 2 2 0 2 

RsaI GTAC 1 113 11 19 19 3 3 3 3 2 2 2 2 2 2 2 4 3 

RseI CAYNNNNRTG 4 62 7 2 2 7 3 3 3 3 3 3 4 4 4 4 4 4 

SacI GAGCTC 0 2 0 1 1 0 1 1 1 1 1 1 1 1 1 1 0 0 

SalI GTCGAC 0 2 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 

SanDI GGGWCCC 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

SatI GCNGC 20 380 31 17 17 42 19 19 19 20 20 20 23 23 23 23 19 37 

ScaI AGTACT 1 5 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 

SchI GAGTC(5/5) 5 61 10 8 8 4 4 4 3 7 7 6 6 6 6 6 6 4 

SdaI CCTGCAGG 0 5 0 1 1 0 1 1 0 1 1 0 0 0 0 0 0 0 

SduI GDGCHC 3 38 3 5 5 10 5 5 6 5 5 6 6 6 6 6 4 8 

SetI ASST 35 592 79 95 95 48 34 34 34 37 37 37 37 37 37 37 39 44 

SexAI ACCWGGT 0 5 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

SfaAI GCGATCGC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 

SfiI GGCCNNNNNGGCC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

SgrAI CRCCGGYG 0 6 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 2 

SgrDI CGTCGACG 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

SgsI GGCGCGCC 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

SmaI CCCGGG 0 3 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 

SmiI ATTTAAAT 2 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 

SmoI CTYRAG 6 17 5 5 5 6 4 4 4 4 4 4 5 5 5 5 4 4 

FauI CCCGC(4/6) 1 90 0 10 10 10 5 5 5 5 5 5 5 5 5 5 1 10 

SrfI GCCCGGGC 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

SsiI CCGC(-3/-1) 28 516 36 42 42 67 34 34 34 32 32 32 36 36 36 36 32 56 

SspI AATATT 3 20 1 6 6 1 1 1 1 2 2 2 2 2 2 2 2 2 

SspDI GGCGCC 0 1 2 1 1 4 1 1 1 0 0 0 0 0 0 0 0 4 

TaaI ACNGT 6 187 15 31 31 14 8 8 8 7 7 7 8 8 8 8 14 20 

TaiI ACGT 5 143 19 22 22 10 5 5 5 8 8 8 8 8 8 8 7 9 

TaqI TCGA 6 121 10 12 12 7 4 4 4 5 5 5 7 7 7 7 10 10 

TasI AATT 15 189 25 64 64 8 7 7 7 10 10 10 12 12 12 12 14 13 

TatI WGTACW 1 24 0 5 5 2 2 2 2 1 1 1 1 1 1 1 1 1 

TauI GCSGC 9 181 17 7 7 21 7 7 7 8 8 8 10 10 10 10 12 22 

Tru1I TTAA 23 195 35 63 63 15 13 13 13 18 18 18 19 19 19 19 16 12 

TscAI CASTG(2/-7) 9 119 6 8 8 12 10 10 10 10 10 10 10 10 10 10 15 13 

TseI GCWGC 11 199 14 10 10 21 12 12 12 12 12 12 13 13 13 13 7 15 

TspDTI ATGAA(11/9) 9 176 18 29 29 10 4 4 4 4 4 4 4 4 4 4 11 11 

TspGWI ACGGA(11/9) 2 107 7 7 7 5 2 2 2 2 2 2 2 2 2 2 7 4 

Van91I CCANNNNNTGG 0 14 2 0 0 2 0 0 0 0 0 0 0 0 0 0 1 4 

VspI ATTAAT 3 17 2 7 7 1 3 3 3 3 3 3 3 3 3 3 2 1 

XagI CCTNNNNNAGG 0 9 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 1 

XapI RAATTY 4 58 7 11 11 1 1 1 1 3 3 3 3 3 3 3 4 3 

XbaI TCTAGA 1 1 0 1 1 0 1 1 1 1 1 1 1 1 1 1 0 1 

XceI RCATGY 2 32 0 6 6 4 3 3 3 2 2 2 1 1 1 1 1 2 

XcmI CCANNNNNNNNNTGG 0 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

XhoI CTCGAG 1 1 1 0 0 0 0 0 0 0 0 0 1 1 1 1 1 0 

XmaJI CCTAGG 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

XmiI GTMKAC 0 9 2 1 1 2 1 1 1 1 1 1 1 1 1 1 1 2 

pACYC177 

pACYC184

Technical Data 

Genome Sizes 

Virus Length, nt Approx. MW, Da 

Bacteriophage X174 5,380 3.5 x 10 6 

Bacteriophage Lambda 48,502 3.1 x 10 7 

Human Immunodeficiency Virus 1 9,181 3.1 x 10 6 

Rous Sarcoma Virus 9,392 3.2 x 10 6 

SARS Coronavirus 29,751 1.0 x 10 7 

Simian Virus 40 (SV 40) 5,224 3.4 x 10 6 

Vaccinia Virus 191,737 1.2 x 10 8 

Variolla (Small pox) Virus 185,578 1.2 x 10 8 

Bacteria 

Agrobacterium tumefaciens C58- 

Cereon 

Length of 

DNA, nt 

Approx. MW, Da 

4.9110 6 3.1910 9 

Aquifex aeolicus VF5 1.5510 6 1.0010 9 

Bacillus subtilis 168 4.2010 6 2.7310 9 

Bacillus halodurans 4.2010 6 2.7310 9 

Bordetella pertussis Tohama I 

NCTC-13251 

4.0710 6 2.6510 9 

Chlamydophila pneumoniae CWL029 1.2310 6 8.0010 8 

Deinococcus radiodurans R1 3.2810 6 2.1310 9 

Escherichia coli K12 4.6410 6 3.0210 9 

Escherichia coli 0157:H7 EDL933 4.1010 6 2.6610 9 

Haemophilus influenzae KW20 1.8310 6 1.1910 9 

Helicobacter pylori 26695 1.6610 6 1.0810 9 

Lactobacillus acidophilus NCFM 1.9910 6 1.2910 9 

Lactococcus lactis IL1403 2.3610 6 1.5310 9 

Mycobacterium leprae TN 3.2710 6 2.1210 9 

Mycobacterium tuberculosis H37Rv 4.4510 6 2.8910 9 

Mycoplasma genitalium G037 5.8010 5 3.7710 8 

Mycoplasma pneumoniae M129 8.1610 5 5.3010 8 

Neisseria meningitidis Z2491 2.1810 6 1.4210 9 

Salmonella typhimurium LT2 SGSC1412 4.8610 6 3.1610 9 

Staphylococcus aureus MW2 2.8210 6 1.8310 9 

Streptomyces coelicolor A3(2) 8.6710 6 5.6410 9 

Streptococcus pneumoniae R6 2.0410 6 1.3310 9 

Streptococcus pyogenes SF370 (M1) 1.8510 6 1.2010 9 

Pseudomonas aeruginosa 6.2610 6 4.0710 9 

Pseudomonas fluorescens Pt-5 7.0710 6 4.6010 9 

Thermus thermophilus HB8 2.1210 6 1.3810 9 

Vibrio cholerae N16961 4.0010 6 2.6010 9 

References 

1. GOLD Genomes online Database, 

for more complete data and updates, see http://www.genomesonline.org/. 

2. Genomes Atlas Database, 

for more complete data and updates, see http://www.cbs.dtu.dk/services/GenomeAtlas/. 

Eukaryotes 

Anopheles gambiae PEST 

(malaria mosquito) 

Archaea 

Length of 

DNA, nt 


Halobacterium sp. NRC-1 2.0110 6 1.3110 9 

Methanosarcina acetivorans C2A 5.7510 6 3.7410 9 

Methanococcus jannaschii DSM2661 1.6610 6 1.0810 9 

Methanosarcina mazei Go1 4.1010 6 2.6610 9 

Methanobacterium 

thermoautotrophicum delta H 

Length of 

DNA, nt 


2.7810 8 1.8010 11 

Arabidopsis thaliana (flowering plant) 1.1510 8 7.4710 10 

Caenorhabditis briggsae 

(soil-dwelling nematode) 

1.0410 8 6.7610 10 

Caenorhabditis elegans (round worm) 1.2110 7 7.8610 9 

Ciona intestinalis (ascidian tadpole) 1.1610 8 7.5410 10 

Drosophila melanogaster (fruit fly) 1.3710 8 8.9010 10 

Gallus gallus (chicken) 1.0510 9 6.8210 11 

Guillardia theta (chromophyte algae) 5.5110 5 3.5810 8 

Homo sapiens (human) 3.1510 9 2.0710 12 

Mus musculus (mouse) ~3.0010 9 ~1.9510 12 

Neurospora crassa OR74A 

(filamentous fungus) 

Saccharomyces cerevisiae S288C 

(budding yeast) 

Schizosaccharomyces pombe 

(fission yeast) 

4.3010 7 2.8010 10 

1.2110 7 7.8610 9 

1.4010 7 9.1010 9 

Pan troglodytes (chimpanzee) 3.0210 9 1.9610 12 

Plasmodium falciparum 3D7 

(human malaria parasite) 

2.2910 7 1.4910 10 

Oryza sativa japonica (rice) 4.2010 8 2.7310 11 

1.7510 6 1.1410 9 

Pyrococcus horikoshii OT3 1.7410 6 1.1310 9 

Pyrococcus abysii GE5 1.7610 6 1.1410 9 

Pyrococcus furiosus DSM3638 1.9110 6 1.2410 9 

Sulfolobus acidocaldarius DSM639 2.2310 6 1.4510 9 

Sulfolobus solfataricus P2 2.9910 6 1.9410 9 

Thermoplasma acidophilum 1.5610 6 1.0110 9 

Calculation the number of copies of a template 

amount of genome DNA (μg) Avogadro constant (mol-1 ) 

Genome copy number = 

length of DNA (bp) 106 650 

This calculation is based on the assumption that the average weight of a base pair (bp) is 650 Daltons. 

Example: Human genome copy number in 1 μg of genome DNA. 

1 (μg) 6.022 10 23 (mol -1 ) 

Genome copy number = = 2.94 10 5 

3.15 10 9 (bp) 10 6 650 


462 

DNA Migration in Agarose and Polyacrylamide Gels 

Recommended agarose gels for electrophoretic separation of DNA fragments. 

Range of Approximate positions of tracking dyes, bp* 

Agarose 

effective Bromophenol blue Xylene cyanol FF 

gel, % 

separation, bp TBE buffer TAE buffer TBE buffer TAE buffer 

0.5 2000-50,000 750 1150 13,000 16,700 

0.6 1000-20,000 540 850 8820 11,600 

0.7 800-12,000 410 660 6400 8500 

0.8 800-10,000 320 530 4830 6500 

0.9 600-10,000 260 440 3770 5140 

1.0 400-8000 220 370 3030 4160 

1.2 300-7000 160 275 2070 2890 

1.5 200-3000 110 190 1300 1840 

2.0 100-2000 65 120 710 1040 

3.0 25-1000 30 60 300 460 

4.0 10-500 18 40 170 260 

5.0 10-300 12 27 105 165 

* Positions of the tracking dyes can be estimated only approximately because the dye front migrates as 

fuzzy band. To use the data above, the following guidelines are recommended: 

 

prepare the gels. 

 

supplied by us (50X TAE Buffer (#B49) and 10X TBE Buffer (#B52), respectively). 

Recommended polyacrylamide gels for electrophoretic separation of DNA fragments (1). 

Polyacrylamide Range of Approximate positions of tracking dyes* 

gel (with BIS at effective 

1:20), % (w/v) separation* 

Bromophenol blue Xylene cyanol FF 

Denaturing gels 

4.0 100-500 nt 50 nt 230 nt 

5.0 70-400 nt 35 nt 130 nt 

6.0 40-300 nt 26 nt 105 nt 

8.0 30-200 nt 19 nt 75 nt 

10.0 20-100 nt 12 nt 55 nt 

15.0 10-50 nt 10 nt 40 nt 

20.0 5-30 nt 8 nt 28 nt 

30.0 1-10 nt 6 nt 20 nt 

Non-denaturing gels 

3.5 100-1000 bp 100 bp 460 bp 

5.0 80-500 bp 65 bp 260 bp 

8.0 60-400 bp 45 bp 160 bp 

12.0 50-200 bp 20 bp 70 bp 

15.0 25-150 bp 15 bp 60 bp 

20.0 5-100 bp 12 bp 45 bp 

* Positions of the tracking dyes can be estimated only approximately because the dye front migrates as 

fuzzy band. 

Tracking dye migration in agarose gels. 

100 kb 

10 kb 

1 kb 

100 bp 

10 bp 

0.5 

Reference 

1. Sambrook, J., et al., Molecular Cloning. A Laboratory 

Manual, Cold Spring Harbor Laboratory, Cold Spring 

Harbor, N.Y., 12.89, 5.42, 2001. 


Bromphenol blue in TBE buffer 

Bromphenol blue in TAE buffer 

Xylene cyanol FF in TBE buffer 

Xylene cyanol FF in TAE buffer 

1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 

Agarose, % 

DNA Base Pairs 

Cytosine ------------------------------------ Guanine 

Thymine ------------------------------------- Adenine 

Codons and Assigned Amino Acids 

First (5’) Second Third (3’) 

U C A G 

U 

C 

A 

G 

Phe 

Phe 

Leu 

Leu 

Leu 

Leu 

Leu 

Leu 

Ile 

Ile 

Ile 

Met (fMet) 

Val 

Val 

Val 

Val** 

* Translation termination codon. 

** Codes for fMet if in the initiator position. 

Ser 

Ser 

Ser 

Ser 

Pro 

Pro 

Pro 

Pro 

Thr 

Thr 

Thr 

Thr 

Ala 

Ala 

Ala 

Ala 

Tyr 

Tyr 

Ter* 

Ter* 

His 

His 

Gln 

Gln 

Asn 

Asn 

Lys 

Lys 

Asp 

Asp 

Glu 

Glu 

Cys 

Cys 

Ter* 

Trp 

Arg 

Arg 

Arg 

Arg 

Ser 

Ser 

Arg 

Arg 

Gly 

Gly 

Gly 

Gly 

Summary of Useful Conversion 

1 Becquerel (Bq) = 1 disintegration per second = 2.7x10-11 Curies (Ci) 

1 Ci = 3.7x1010 Bq = 37 GBq = 2.22x1012 disintegrations per minute (dpm) 

1 mCi = 37 MBq = 2.22x109 dpm 

1 μCi = 37 kBq = 2.22x106 dpm 

1 GBq = 27 mCi 

1 MBq = 27 μCi 

1 kBq = 27 nCi 

U 

C 

A 

G 

U 

C 

A 

G 

U 

C 

A 

G 

U 

C 

A 

G 

Physical Properties of Some Common Radioisotopes 

Radioisotope Half-life Specific activity, MBq/mmol 

32P 14.3 days 7 up to 10 

33P 25.4 days 7 up to 10 

35S 87.4 days 7 up to 10 

131I 8.04 days 5 up to 10 

125I 60 days 7 up to 10 

14C 5730 years 3 up to 10 

3H 12.43 years 6 

up to 10

Amino Acids 

Nonpolar (hydrophobic) 

Amino acid Structure 

Alanine 

Proline 

Valine 

Leucine 

Isoleucine 

Methionine 

Phenylalanine 

Tryptophan 

Polar, uncharged 


Glycine 

Serine 

Threonine 

Cysteine 

Asparagine 

Glutamine 

Tyrosine 

Basic 


Lysine 

Histidine 

Arginine 

Amino acid 

Abbreviation 

3-letter 1-letter 

Acidic 


Aspartate 

Glutamate 

M r, Da 

pK 1 

(-COOH – ) 

pKa values 

pK 2 

(-NH 3 + ) 

pK R 

(R group) 

Nonpolar (hydrophobic) 

Alanine Ala A 89.09 2.34 9.69 6.01 

Proline Pro P 115.13 1.99 10.96 6.48 

Valine Val V 117.15 2.32 9.62 5.97 

Leucine Leu L 131.17 2.36 9.60 5.98 

Isoleucine Ile I 131.17 2.36 9.68 6.02 

Methionine Met M 149.21 2.28 9.21 5.74 

Phenylalanine Phe F 165.19 1.83 9.13 5.48 

Tryptophan Trp W 204.23 2.38 9.39 5.89 

Polar, uncharged 

Glycine Gly G 75.07 2.34 9.60 5.97 

Serine Ser S 105.09 2.21 9.15 5.68 

Threonine Thr T 119.12 2.11 9.62 5.87 

Cysteine Cys C 121.16 1.96 10.28 8.18 5.07 

Asparagine Asn N 132.12 2.02 8.80 5.41 

Glutamine Gln Q 146.15 2.17 9.13 5.65 

Tyrosine Tyr Y 181.19 

Basic 

2.20 9.11 10.07 5.66 

Lysine Lys K 146.19 2.18 8.95 10.53 9.74 

Histidine His H 155.16 1.82 9.17 6.00 7.59 

Arginine Arg R 174.20 

Acidic 

2.17 9.04 12.48 10.76 

Aspartate Asp D 133.10 1.88 9.60 3.65 2.77 

Glutamate Glu E 147.13 2.19 9.67 4.25 3.22 

pI 


464 

Concentrations of Acids and Bases 

Substance Formula MW Moles/liter* Grams/liter 

* With some acids and bases, stock solutions of different molarity/normality are in common use. 


% 

by weight 

Specific 

gravity 

ml/liter to prepare 

1 M solution 

Acetic acid, glacial CH3COOH 60.05 17.4 1045 99.5 1.05 57.5 

Acetic acid CH3COOH 60.05 6.27 376 36 1.045 159.5 

Formic acid HCOOH 46.02 23.4 1080 90 1.20 42.7 

Hydrochloric acid HCl 36.5 11.6 424 36 1.18 86.2 

Nitric acid HNO3 63.02 15.99 1008 71 1.42 62.5 

Perchloric acid HClO4 100.5 11.65 1172 70 1.67 85.8 

Phosphoric acid H3PO4 97.9 14.8 1445 85 1.70 67.7 

Sulfuric acid H2SO4 98.1 18.0 1766 96 1.84 55.6 

Ammonium hydroxide NH4OH 35.0 14.8 251 28 0.898 67.6 

Potassium hydroxide KOH 56.1 13.5 757 50 1.52 74.1 

Sodium hydroxide NaOH 40.0 19.1 763 50 1.53 52.4 

Physical Constants of the Nucleoside 

Triphosphates and Related Compounds 

(see section “Nucleotides & Primers” for ordering information) 

Compound 

MW, 

Da (acid form) 

molar absorption coefficient (absorbance at max for 1M solution at pH 7.0) 

* determined at pH 7.0 

** determined at pH 10.0 

Conversion formula 

C = A / x 10 3 

C – mM concentration of compounds 

A – observed absorbance at max (nm) 

– molar absorption coefficient (M -1 x cm -1 ) 

max*, 

nm 

, 

M -1 x cm -1 

ATP 507 259 15,400 

CTP 483 271 9000 

GTP 523 253 13,700 

UTP 484 262 10,000 

dATP 491 259 15,200 

dCTP 467 271 9300 

dGTP 507 253 13,700 

dTTP 482 267 9600 

ddATP 475 261 15,200 

ddCTP 451 271 9300 

ddGTP 491 253 13,600 

ddTTP 466 267 9600 

dm 6 ATP 507 265 15,400 

dm 4 CTP 481 274 13,600 

dm 5 CTP 481 279 8770 

Aminoallyl-dUTP 523 

Biotin-11-dUTP 862 

240 

290 

240 

289 

11,900 

7800 

10,700 

7100 

NAD 664 260 18,000 

NADH 665 338** 6200 

NADP 743 260 18,000 

NADPH 745 260 18,000 

SI Unit Prefixes 

Prefix Symbol Multiple 

yotta Y 10 24 

zetta Z 10 21 

exa E 10 18 

peta P 10 15 

tera T 10 12 

giga G 10 9 

mega M 10 6 

kilo k 10 3 

hecto h 10 2 

deka da 10 1 

deci d 10 -1 

centi c 10 -2 

milli m 10 -3 

micro μ 10 -6 

nano n 10 -9 

pico p 10 -12 

femto f 10 -15 

atto a 10 -18 

zepto z 10 -21 

yocto y 10 -24

Common Conversions of Nucleic Acids 

Estimation of ends (3’ or 5’) concentration 

Circular DNA 

pmol ends = pmol DNA x number of cuts x 2 

Linear DNA 

pmol ends = pmol DNA x (number of cuts x 2 + 2) 

Phage/Plasmid DNA pmol ends (1 μg) 

1000 bp DNA 3.04 

linear pUC18/19 DNA 1.14 

linear pBR322 DNA 0.7 

linear SV40 DNA 0.58 

linear X174 DNA 0.56 

linear M13mp18/19 DNA 0.42 

phage DNA 0.06 

Spectrophotometric Conversions 

NA A260 μg/ml mM (in nucleotides) 

dsDNA 1 50 0.15 

ssDNA 1 33 0.1 

ssRNA 1 40 0.12 

dsDNA 6.7 335 1 

ssDNA 10.0 330 1 

ssRNA 8.3 332 1 

The average MW of a deoxyribonucleotide base = 333 Da 

The average MW of a ribonucleotide base = 340 Da 

The average MW of a deoxyribonucleotide base pair = 650 Da 

Phage/Plasmid DNA 

Molar Conversions 

bp 

μg 

Quantity 

pmol 

DNA 1000 

1 

0.66 

1.52 

1 

pUC18/19 DNA 2686 

1 

1.77 

0.57 

1 

pBR322 DNA 4361 

1 

2.88 

0.35 

1 

SV40 DNA 5243 

1 

3.46 

0.29 

1 

X174 DNA 5386 

1 

3.54 

0.28 

1 

M13mp18/19 DNA 7250 

1 

4.78 

0.21 

1 

phage DNA 48502 

1 

32.01 

0.03 

11 

DNA/Protein Conversions 

1 kb of DNA = 333 amino acid 37 kDa 

10 kDa protein 0.27 kb DNA 




Common Conversions of Oligonucleotides 

Common conversions of Oligonucleotides 


MW = 333 x N 

Concentration of Oligonucleotides 

C (μM or pmol/μl) = A 260 / (0.01 x N) 

C (ng/ml) = (A 260 x MW) / (0.01 x N) 

MW – molecular weight 

A 260 – absorbance at 260 nm 

N – number of bases 

Melting Temperature of Duplex DNA 

and Oligonucleotides 

For Duplex Oligonucleotide shorter than 25 bp, 

“The Wallace Rule” (2) 

Tm (in °C) = 2(A+T) + 4(C+G), where 

(A+T) – the sum of the A and T residues in the oligonucleotide and 

(G+C) – the sum of G and C residues in the oligonucleotide. 

Presence of m5C in oligonucleotide increases melting temperature of 

duplex. Presence of m4C or m6A decreases melting temperature (3). 

For Duplex DNA,

466 

Temperature Dependence of the pH for Commonly Used Buffers 

Buffer System Chemical Name pKa/20°C pKa/10°C MES 4-morpholineethanesulfonic acid 6.15 -0.110 

ADA [(carbamoylmethyl)imino]diacetic acid 6.60 -0.110 

PIPES 1,4-piperazinediethanesulfonic acid 6.80 -0.085 

ACES 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid 6.90 -0.200 

BES 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid 7.15 -0.160 

MOPS 4-morpholinepropanesulfonic acid 7.20 -0.013 

TES 

2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}- 

1-propanesulfonic acid 

7.50 -0.200 

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 7.55 -0.140 

TRICINE N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 8.15 -0.210 

TRIS 2-amino-2-hydroxymethylpropane-1,3-diol 8.30 -0.310 

BICINE N,N-bis(2-hydroxyethyl)glycine 8.35 -0.180 

GLYCYLGLYCINE 2-(2-aminoacetyl)aminoacetic acid 8.40 -0.280 

Decay Factors for Calculating the Amount of Radioactivity 

32P, (half-life – 14.3 days) 

Days 

0 12 24 36 

Hours 

48 60 72 84 

0 1.000 0.976 0.953 0.930 0.908 0.886 0.865 0.844 

4 0.824 0.804 0.785 0.766 0.748 0.730 0.712 0.695 

8 0.679 0.662 0.646 0.631 0.616 0.601 0.587 0.573 

12 0.559 0.546 0.533 0.520 0.507 0.495 0.483 0.472 

16 0.460 0.449 0.439 0.428 0.418 0.408 0.398 0.389 

20 0.379 0.370 0.361 0.353 0.344 0.336 0.328 0.320 

24 0.312 0.305 0.298 0.291 0.284 0.277 0.270 0.264 

28 0.257 0.251 0.245 0.239 0.234 0.228 0.223 0.217 

32 0.212 0.207 0.202 0.197 0.192 0.188 0.183 0.179 

36 0.175 0.170 0.166 0.162 0.159 0.155 0.151 0.147 

40 0.144 0.140 0.137 0.134 0.131 0.127 0.124 0.121 

44 0.119 0.116 0.113 0.110 0.108 0.105 0.102 0.100 

48 0.098 0.095 0.093 0.091 0.089 0.086 0.084 0.082 

52 0.080 0.078 0.077 0.075 0.073 0.071 0.070 0.068 

35S, (half-life – 84.4 days) 

Weeks 

0 1 2 

Days 

3 4 5 6 

0 1.000 0.992 0.984 0.976 0.969 0.961 0.954 

1 0.946 0.939 0.931 0.924 0.916 0.909 0.902 

2 0.895 0.888 0.881 0.874 0.867 0.860 0.853 

3 0.847 0.840 0.833 0.827 0.820 0.814 0.807 

4 0.801 0.795 0.788 0.782 0.776 0.770 0.764 

5 0.758 0.752 0.746 0.740 0.734 0.728 0.722 

6 0.717 0.711 0.705 0.700 0.694 0.689 0.683 

7 0.678 0.673 0.667 0.662 0.657 0.652 0.646 

8 0.641 0.636 0.631 0.626 0.621 0.616 0.612 

9 0.607 0.602 0.597 0.592 0.588 0.583 0.579 

10 0.574 0.569 0.565 0.560 0.556 0.552 0.547 

11 0.543 0.539 0.534 0.530 0.526 0.522 0.518 

12 0.514 0.510 0.506 0.502 0.498 0.494 0.490 

33P, (half-life – 25.4 days) 

Days 

0 1 1 3 4 

Days 

5 6 7 8 8 

0 1.000 0.973 0.947 0.921 0.897 0.872 0.849 0.826 0.804 0.782 

10 0.761 0.741 0.721 0.701 0.683 0.664 0.646 0.629 0.612 0.595 

20 0.579 0.564 0.549 0.534 0.520 0.506 0.492 0.479 0.466 0.453 

30 0.441 0.429 0.418 0.406 0.395 0.385 0.374 0.364 0.355 0.345 

40 0.336 0.327 0.318 0.309 0.301 0.293 0.285 0.277 0.270 0.263 

50 0.256 0.249 0.242 0.236 0.229 0.223 0.217 0.211 0.205 0.200 

60 0.195 0.189 0.184 0.179 0.174 0.170 0.165 0.161 0.156 0.152 

70 0.148 0.144 0.140 0.136 0.133 0.129 0.126 0.122 0.119 0.116 

80 0.113 0.110 0.107 0.104 0.101 0.098 0.096 0.093 0.091 0.088 

90 0.086 0.084 0.081 0.079 0.077 0.075 0.073 0.071 0.069 0.067 

100 0.065 0.064 0.062 0.060 0.059 0.057 0.055 0.054 0.053 0.051 

110 0.050 0.048 0.047 0.046 0.045 0.043 0.042 0.041 0.040 0.039 

120 0.038 0.037 0.036 0.035 0.034 0.033 0.032 0.031 0.030 0.030 


Commonly Used Media, Stock Solutions and Buffers 

Growth Media (1) 

LB Medium, per liter: Final 1X concentration: 

Tryptone 10 g 1.0% (w/v) 

Yeast extract 5 g 0.5% (w/v) 

NaCl 10 g 1.0% (w/v) 

H 2O to 1 liter 

Adjust pH to 7.0. 

Low Salt LB Medium, per liter: Final 1X concentration: 



NaCl 5 g 0.5% (w/v) 


Adjust pH to 7.0. 

Terrific Broth Medium, per liter: Final 1X concentration: 



Glycerol 4 ml 0.4% (w/v) 

H 2O to 900 ml 

Autoclave, cool to 60°C or less before adding 100 ml of filter sterilized 10X TB 

phosphate (0.17 M KH 2PO 4, 0.72 M K 2HPO 4). 

SOB Medium, per liter Final 1X concentration: 



NaCl 0.5 g 0.05% (w/v) 

250 mM KCl 10 ml 2.5 mM 


Adjust pH to 7.0 and add H 2O to 990 ml. 

Autoclave, cool to room temperature and just prior to use add 10 ml 

1 M MgCl 2 (sterile solution). 10 mM 

SOC Medium, per liter: 

SOB Medium (1 liter) with the addition of 20 ml filter sterilized 

1 M glucose. 

M9 Minimal Medium, per liter: Final 1X concentration: 

5X M9 salts 200 ml 

1 M MgSO 4 1 ml 1 mM 

20% glucose 10 ml 0.2% (v/v) 

1M CaCl 2 0.1 ml 0.1 mM 

Sterile H 2O to 1 liter 

5X M9 Salts, per liter: Final 1X concentration: 

Na 2HPO 4x7H 2O 64 g 47.8 mM 

KH 2PO 4 15 g 22 mM 

NaCl 2.5 g 8.6 mM 

NH 4Cl 5 g 18.7 mM 

Additives Final 1X concentration: 

Antibiotics (if required): 

Ampicillin 50 μg/ml 

Chloramphenicol 20 μg/ml 

Kanamycin 30 μg/ml 

Tetracycline 12 μg/ml 

Galactosides (if required): 

X-Gal 20 μg/ml 

IPTG 0.1 mM 

Media containing agar or agarose, per liter: 

Agar (for plates) 15 g 1.5% (w/v) 

Agar (for top agar) 7 g 0.7% (w/v) 

Agarose (for plates) 15 g 1.5% (w/v) 

Agarose (for top agarose) 7 g 0.7% (w/v) 

Stock Solutions 

10 M Ammonium Acetate: 

Ammonium acetate 385.4 g 

H2O to 500 ml 

1 M CaCl2: CaCl2x6H2O 219.1 g 

H2O to 1 liter 



468 

100X Denhardt Solution: Final concentration: 

Ficoll 400 10 g 0.02% (w/v) 

Polyvinylpyrrolidone 10 g 0.02% (w/v) 

Bovine serum albumin 10 g 0.02% (w/v) 


Filter sterilize and store at -20°C in 25 ml aliquots 

1 M Dithiothreitol (DTT): 

DTT 15.45 g 

H 2O to100 ml 

Store at -20°C 

0.5 M Ethylenediamine Tetraacetic Acid (EDTA) (pH 8.0): 

Na 2EDTAx2H 2O 186.1 g 


Adjust pH to 8.0 with 10 M NaOH (~50 ml) 


10 mg/ml Ethidium Bromide: 

Ethidium bromide 0.2 g 

H 2O to 20 ml 

Mix well and store at 4°C in dark. 

CAUTION: Ethidium bromide is a mutagen. Wear gloves when working with 

solutions and a mask when weighing the powder. 

1 M KCl: 

KCl 74.6 g 


1 M MgCl 2: 

MgCl 2x6H 2O 20.3 g 


1 M MgSO 4: 

MgSO 4x7H 2O 24.6g 

H 2O to 100ml 

5M NaCl: 

NaCl 292.2 g 


10 M NaOH: 

NaOH 400.0 g 


1 M Tris-HCl [tris(hydroxymethyl)aminomethane]: 

Tris base 121.1 g 


Adjust to desired pH with concentrated HCl. 

Mix and add H 2O to 1 liter 

3 M Sodium Acetate (pH 5.2 or 7.0) (2): 

Sodium acetate x 3H 2O 408.1 g 


Adjust the pH to 7.0 with acetic acid or adjust the pH to 5.2 with glacial acetic acid. 


Buffers (2) 

10X Stock Phosphate-buffered Saline (PBS), per liter: Final 1X concentration: 

NaCl 80.0 g 137 mM 

KCl 2.0 g 2.7 mM 

Na2HPO4 14.4 g 10 mM 

KH2PO4 2.7 g 2 mM 

H2O Adjust to pH 7.4 with concentrated HCl. 

to 800 ml 


20X SSC, per liter: Final 1X concentration: 

NaCl 175.3 g 150 mM 

Na3citrate x 2H2O 88.2 g 15 mM 

H2O Adjust pH to 7.0 with 1M HCl 

to 800 ml 




20X SSPE, per liter: Final 1X concentration: 

NaCl 175.3 g 150 mM 

NaH 2PO 4xH 2O 27.6 g 10 mM 

Na 2EDTA 7.4 g 1 mM 


Adjust pH to 7.4 with 10 M NaOH 


5X Tris-glycine, Native Electrophoresis Buffer, per liter: Final 1X concentration: 

Tris base 15.1 g 25 mM 

Glycine 72.0 g 192 mM 


The pH of diluted solution is pH 8.3. 

5X Tris-glycine-SDS Electrophoresis Buffer, per liter: Final 1X concentration: 

Tris base 15.1 g 25 mM 

Glycine 72.0 g 192 mM 

SDS 5.0 g 0.1% (w/v) 



5X Tris-tricine-SDS Electrophoresis Buffer, per liter: Final 1X concentration: 

Tris base 60.6 g 0.1 M 

Tricine 89.6 g 0.1 M 

SDS 5.0 g 0.1% (w/v) 



50X TAE (Tris-acetate-EDTA) Electrophoresis Buffer, per liter: Final 1X concentration: 

Tris base 242 g 40 mM 

Glacial acetic acid 57.1 ml 20 mM 

0.5 M EDTA (pH 8.0) 100 ml 1 mM 


The pH of diluted solution is ~8.5 

10X TBE (Tris-borate-EDTA) Electrophoresis Buffer, per liter: Final 1X concentration: 


Boric acid 55 g 89 mM 

0.5 M EDTA (pH 8.0) 40 ml 2 mM 


10X TPE (Tris-phosphate-EDTA) Electrophoresis Buffer, per liter: Final 1X concentration: 


Phosphoric acid (85%) 15.5 ml 27 mM 

0.5 M EDTA (pH 8.0) 40 ml 2 mM 


TE (Tris-EDTA) Buffer, pH 7.4, 7.6 or 8.0, per liter: Final 1X concentration: 

1 M Tris, pH 7.4, 7.6, 8.0 10 ml 10 mM 

0.5 M EDTA (pH 8.0) 2 ml 1 mM 


Molarity 

M (mol/l) = 

Expressing Concentrations of Reagents 

number of moles solute 

number of liters of solution 

Percent composition 

weight of solute (g) 

Concentration (%) = x 100 

total weight of solution (g) 

Percent composition (weight/volume) 

weight of solute (g) 

Concentration (% w/v) = x 100 

total weight of solution (ml) 

Percent composition (volume/volume) 

weight of solute (ml) 

Concentration (% v/v) = x 100 

total weight of solution (ml) 

References 

1. Atlas, R.M., Handbook of Microbiological Media, second edition, Ed. Parks, L.C., CRC 

Press, N.Y., 1997. 

2. Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, second edition, Cold Spring 

Harbor Laboratory Press, Cold Spring Harbor, NY, A1.2-A2.12, 2001. 



470 

Periodic Table of the Elements 

IA VIIIA 

1 

H 

Hydrogen 

1.0079 IIA IIIA IVA VA VIA VIIA 

2 

He 

Helium 

4.0026 

3 

Li 

Lithium 

6.941 

4 

Be 

Beryllium 

9.0122 

5 

B 

Boron 

10.811 

6 

C 

Carbon 

12.011 

7 

N 

Nitrogen 

14.007 

8 

O 

Oxygen 

15.999 

9 

F 

Fluorine 

18.998 

10 

Ne 

Neon 

20.180 

11 

Na 

Sodium 

22.990 

12 

Mg 

Magnesium 

24.305 IIIB IVB VB VIB VIIB VIIB VIIB VIII IB IIB 

13 

Al 

Aluminium 

26.982 

14 

Si 

Silicon 

28.086 

15 

P 

Phosphorus 

30.974 

16 

S 

Sulfur 

32.066 

17 

Cl 

Chlorine 

35.453 

18 

Ar 

Argon 

39.948 

19 

K 

Potassium 

39.098 

20 

Ca 

Calcium 

40.078 

21 

Sc 

Scandium 

44.956 

22 

Ti 

Titanium 

47.897 

23 

V 

Vanadium 

50.942 

24 

Cr 

Cromium 

51.996 

25 

Mn 

Manganese 

54.938 

26 

Fe 

Iron 

55.847 

27 

Co 

Cobalt 

58.933 

28 

Ni 

Nickel 

58.693 

29 

Cu 

Copper 

63.546 

30 

Zn 

Zinc 

65.39 

31 

Ga 

Gallium 

69.723 

32 

Ge 

Germanium 

72.61 

33 

As 

Arsenic 

74.922 

34 

Se 

Selenium 

78.96 

35 

Br 

Bromine 

79.904 

36 

Kr 

Krypton 

83.80 

37 

Rb 

Rubidium 

85.468 

38 

Sr 

Strontium 

87.62 

39 

Y 

Yttrium 

88.906 

40 

Zr 

Zirconium 

91.224 

41 

Nb 

Niobium 

92.906 

42 

Mo 

Molybdenum 

95.94 

43 

Tc 

Technetium 

[98] 

44 

Ru 

Ruthenium 

101.07 

45 

Rh 

Rhodium 

102.91 

46 

Pd 

Palladium 

105.42 

47 

Ag 

Silver 

107.87 

48 

Cd 

Cadmium 

112.41 

49 

In 

Indium 

114.82 

50 

Sn 

Tin 

118.71 

51 

Sb 

Antimony 

121.75 

52 

Te 

Tellurium 

127.60 

53 

I 

Iodine 

126.90 

54 

Xe 

Xenon 

131.29 

55 

Cs 

Cesium 

132.91 

56 

Ba 

Barium 

137.33 

57-71 

* 

72 

Hf 

Hafnium 

178.49 

73 

Ta 

Tantalum 

180.95 

74 

W 

Tungsten 

183.85 

75 

Re 

Rhenium 

186.21 

76 

Os 

Osmium 

190.23 

77 

Ir 

Iridium 

192.22 

78 

Pt 

Platinum 

195.08 

79 

Au 

Gold 

196.97 

80 

Hg 

Mercury 

200.59 

81 

Tl 

Thalium 

204.38 

82 

Pb 

Lead 

207.2 

83 

Bi 

Bismuth 

208.98 

84 

Po 

Polonium 

[209] 

85 

At 

Astatine 

[210] 

86 

Rn 

Radon 

[222] 

87 

Fr 

Francium 

[223] 

88 

Ra 

Radium 

[226.03] 

89-103 

* * 

104 

Rf 

Rutherfordium 

[261] 

105 

Db 

Dubnium 

[262] 

106 

Sg 

Seaborgium 

[263] 

107 

Bh 

Bohrium 

[264] 

108 

Hs 

Hassium 

[269] 

109 

Mt 

Meitnerium 

[268] 

110 

Uun 

Ununnilium 

[271] 

57 

La 

Lanthanum 

138.91 

58 

Ce 

Cerium 

140.12 

59 

Pr 

Praseodymium 

140.91 

60 

Nd 

Neodymium 

144.24 

61 

Pm 

Promethium 

[145] 

62 

Sm 

Samarium 

150.36 

63 

Eu 

Europium 

151.97 

64 

Gd 

Gadolinium 

157.25 

65 

Tb 

Terbium 

158.93 

66 

Dy 

Dysprosium 

162.50 

67 

Ho 

Holmium 

164.93 

68 

Er 

Erbium 

167.26 

69 

Tm 

Thulium 

168.93 

70 

Yb 

Ytterbium 

173.04 

71 

Lu 

Lutetium 

174.97 

89 

Ac 

Actinium 

[227] 

90 

Th 

Thorium 

232.04 

91 

Pa 

Protactinium 

231.04 

92 

U 

Uranium 

238.03 

93 

Np 

Neptunium 

237.05 

94 

Pu 

Plutonium 

[244] 

95 

Am 

Americium 

[243] 

96 

Cm 

Curium 

[247] 

97 

Bk 

Berkelium 

[247] 

98 

Cf 

Californium 

[251] 

99 

Es 

Einsteinium 

[252] 

100 

Fm 

Fermium 

[257] 

101 

Md 

Mendelevium 

[258] 

102 

No 

Nobelium 

[259] 

103 

Lr 

Lawrencium 

[262] 

* Lanthanide series 

** Actinide series 

Alkali metals 

Alkaline earth metals 

Transition metals 

Poor metals 

Solid 

Liquid 

Gas 

Synthetic 

Br 

H 

Tc 

C 

Nonmetals 

Noble gases 

Lanthanide series 

Actinide series 

+1 

-1 

+1 

+1 

+1 

+1 

+1 

+1 +2 

+2 

+2 

+2 

+2 

+2 

+3 

+3 

+3 

+4 

+4 

+4 

+4 +5 

+3 

+4 

+5 

+3 

+5 

+2 

+3 

+4 

+5 

+2 

+3 

+6 

+2 

+3 

+4 

+5 

+6 

+2 

+3 

+4 

+5 

+6 

+2 

+3 

+4 

+5 

+6 

+7 

+2 

+4 

+7 

+2 

+3 

+4 

+6 

+7 

+3 

+4 

+6 

+8 

+2 

+3 

+4 

+6 

+8 

+2 

+3 

+6 

+2 

+3 

+2 

+3 

+4 

+5 

+2 

+3 

+4 

+6 

+2 

+3 

+2 

+4 

+2 

+4 

+6 

+1 

+2 

+3 

+1 

+2 

+3 

+1 

+3 

+2 

+2 

+1 

+2 

+3 

+3 

+3 

+1 

+3 

+1 

+3 

+2 

+4 

+4 

+2 

+4 

+2 

+4 

+2 

+4 

-3 

+2 

+3 

+4 

+5 

-3 

+3 

+5 

-3 

+3 

+5 

-3 

+3 

+5 

+3 

+5 

-1 

-2 

-2 

+2 

+4 

+6 

-2 

+4 

+6 

-2 

+4 

+6 

+2 

+4 

+6 

-1 

-1 

+1 

+3 

+5 

+7 

-1 

+1 

+3 

+5 

-1 

+1 

+5 

+7 

-1 

+1 

+3 

+5 

+7 

- 

- 

- 

+2 

+2 

+4 

+6 

+8 

+2 

+3 

+3 

+3 

+4 

+4 

+3 

+4 

+4 

+5 

+3 

+3 

+4 

+5 

+6 

+3 

+3 

+4 

+5 

+6 

+2 

+3 

+3 

+4 

+5 

+6 

+2 

+3 

+3 

+4 

+5 

+6 

+3 

+3 

+4 

+3 

+4 

+2 

+3 

+4 

+3 

+2 

+3 

+4 

+3 

+2 

+3 

+3 

+2 

+3 

+3 

+2 

+3 

+2 

+3 

+2 

+3 

+3 

+3 

6 

C 

Carbon 

12.011 

Oxidation degree 

Symbol 

Name 

Atomic weight 

IVA 

Group classification 

+2 

+4 

Atomic number

Commonly Used Abbreviations 

A 

A adenine or adenosine; one-letter code for alanine 

A 260 absorbance at 260 nm 

ACES N-(2-acetamido)-2-aminoethanesulfonic acid 

Ad-2 adenovirus-2 

ADA N-(2-acetamido)-2-iminodiacetic acid 

ADP adenosine 5’-diphosphate 

AMP adenosine monophosphate 

AMV Avian Myeloblastosis Virus 

AP alkaline phosphatase 

APS ammonium persulfate 

aRNA antisense RNA 

ATP adenosine 5’-triphosphate 

B 

BCIP 5-bromo-4-chloro-3-indolyl phosphate 

BES N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid 

BGG bovine gamma globulin 

BICINE N,N-bis(2-hydroxyethyl)glycine 

Bio-dNTP biotin-deoxynucleoside triphosphate 

BME -mercaptoethanol; 2-mercaptoethanol 

bp base pair 

Bq Becquerel 

BSA bovine serum albumin 

B/W blue/white cloning 

C 

C cytosine or cytidine; one-letter code for cysteine 

CA casamino-acids 

CAPS N-cyclohexyl-3-aminopropanesulfonic acid 

cDNA complementary deoxyribonucleic acid 

CEU cohesive end ligation unit 

Ci Curie 

CIAP calf intestinal alkaline phosphatase 

CMP cytidine monophosphate 

cpm counts per minute 

CTP cytidine 5’-triphosphate 

D 

Da Dalton 

dAMP deoxyadenosine monophosphate 

dATP deoxyadenosine triphosphate 

dCTP deoxycytidine triphosphate 

ddATP dideoxyadenosine triphosphate 

ddCTP dideoxycytidine triphosphate 

ddGTP dideoxyguanosine triphosphate 

ddNTP dideoxynucleoside triphosphate 

ddTTP dideoxythymidine triphosphate 

DE-81 Whatman diethylaminoethyl cellulose paper 

DEPC diethyl pyrocarbonate 

DIG digoxigenin 

dITP deoxyinosine triphosphate 

dGTP deoxyguanosine triphosphate 

DMSO dimethyl sulfoxide 

DNA deoxyribonucleic acid 

DNase deoxyribonuclease 

dNMP deoxyribonucleoside monophosphate 

dNTP deoxynucleoside triphosphate 

dpm disintegrations per minute 

ds double-stranded 

DTT dithiothreitol 

dTTP deoxythymidine triphosphate 

dUTP deoxyuridine triphosphate 

E 

EDTA ethylenediaminetetraacetic acid 

EGTA ethylene glycol tetraacetic acid 

ELISA enzyme-linked immunosorbent assay 

EMBL European Molecular Biology Laboratory 

EMSA electrophoretic mobility shift assay 

Endo endodeoxyribonuclease assay 

endo endonuclease 



472 

exo exonuclease 

Exo 5’ and 3’- exodeoxyribonuclease assay 

F 

FDU fast digest unit 

FOA (5-FOA) 5-fluoroorotic acid 

G 

G guanine or guanosine; one-letter code for glycine 

Gal D-galactose 

GMP guanosine monophosphate 

GMPD glycosylase mediated polymorphism detection 

GUS -D-glucuronidase 

GTP guanosine 5’-triphosphate 

GQ genetic quality 

H 

HC high concentration 

HEPES N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) 

HRP horseradish peroxidase 

HPLC high-performance liquid chromatography 

I 

IEF isoelectric focussing 

IPTG isopropyl--D-thiogalactopyranoside 

K 

kb kilobase 

kDa kiloDalton 

L 

LAMP loop-mediated isothermal amplification 

LB Luria Bertani media 

LC low concentration 

LE low electroendosmosis 

LM low melting point 

LO labeled oligonucleotide 

M 

MCS multiple cloning site 

MES 2-(N-morpholino)ethanesulfonic acid 

MDA multiple displacement amplification 

M-MuLV Moloney Murine Leukemia Virus 

MOPS 3-(N-morpholino)propanesulfonic acid 

mRNA messenger ribonucleic acid 

MW molecular weight 

N 

NAD nicotinamide adenine dinucleotide 

NADH nicotinamide adenine dinucleotide, reduced form 

NADP nicotinamide adenine dinucleotide phosphate 

NADPH nicotinamide adenine dinucleotide phosphate, reduced form 

NBT nitro blue tetrazolium 

NDP nucleoside diphosphate 

NMP ribonucleoside monophosphate 

NR not recommended 

NP-40 Nonidet P-40 (detergent) 

nt nucleotide 

NTC no-template control 

NTP nucleoside triphosphate 

O 

oligo(A) oligoadenylic acid 

oligo(dT) oligodeoxythymidylic acid 

OMP orotidine monophosphate 

P 

P i inorganic phosphate 

PAAG polyacrylamide gel 

PAGE polyacrylamide-gel electrophoresis 

PBS phosphate-buffered saline 

PCR polymerase chain reaction 

PEI polyethylenimine 

PEG polyethylene glycol 

PIPES piperazine-N,N’-bis(2-ethanesulfonic acid) 

PNK polynucleotide kinase 

pNPP 4-nitrophenyl phosphate; para-nitrophenyl phosphate 

poly(A) polyadenylic acid 

poly(A) + polyadenylated (mRNA) 

poly(dA-dT) poly (deoxyadenylic acid – deoxythymidylic acid) 



poly(dT) polydeoxythymidylic acid 

PP i inorganic pyrophosphate 

PVDF polyvinylidene fluoride 

Q 

QCA quality control assay 

qPCR quantitative polymerase chain reaction 

R 

RACE Rapid Amplification of cDNA Ends 

RCA rolling circle amplification 

RDA random displacement amplification 

RE restriction endonuclease 

REase restriction endonuclease 

RNA ribonucleic acid 

RNase ribonuclease 

R-M restriction-modification 

rRNA ribosomal ribonucleic acid 

RT reverse transcriptase 

RT-PCR reverse transcription polymerase chain reaction 

RT-qPCR reverse transcription – quantitative polymerase chain reaction 

S 

SAM S-adenosylmethionine 

SDA strand displacement amplification 

SDS sodium dodecyl sulfate 

siRNA small interfering ribonucleic acid 

SNP single nucleotide polymorphism 

ss single-stranded 

SSC sodium chloride/sodium citrate (buffer) 

SSCP single-strand conformation polymorphism 

SSPE sodium chloride/sodium phosphate/EDTA (buffer) 

STR short tandem repeat 

T 

T thymine or thymidine; one-letter code for threonine 

TAE Tris/acetate/EDTA (buffer) 

TBE Tris/borate/EDTA (buffer) 

TdT terminal deoxynucleotidyl transferase 

TE Tris/EDTA (buffer) 

TEMED N,N,N’,N’-tetramethylethylenediamine 

TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid 

TLC thin layer chromatography 

Tm melting temperature 

TMB 3,3’, 5,5’-tetramethylbenzidine 

TRICINE N-tris(hydroxymethyl)methylglycine 

Tris tris(hydroxymethyl)aminomethane 

tRNA transfer ribonucleic acid 

TPE Tris/phosphate/EDTA (buffer) 

TX-100 triton X-100 

T4 T4 polynucleotide kinase 

U 

U uracil or uridine 

u unit 

UDG uracil-DNA glycosylase 

UMP uracil monophosphate 

UTP uridine 5’-triphosphate 

UV ultraviolet 

V 

v/v volume/volume 

W 

WGA whole genome amplification 

w/v weight/volume 

X 

X-Gal 5-bromo-4-chloro-3-indolyl--D-galactopyranoside 

X-Gluc 5-bromo-4-chloro-3-indolyl--D-glucuronic acid 


474 


INDICES 

Numerical Index .............................................................................................................. 475 

Alphabetical Index .......................................................................................................... 482 

Protocol Index .................................................................................................................. 494

Numerical Index 

Cat. # 

B 

Product name Size Page 

B02 10X Buffer Cfr9I 1 ml 160 

B04 10X Buffer Cfr10I 1 ml 160 

B12 10X Buffer EcoRI 5x1 ml 160 

B13 10X Buffer Bsp143I 1 ml 160 

B14 Bovine Serum Albumin (BSA) 5 mg 422 

B19 Dilution Buffer for Restriction Enzymes 5x1 ml 160 

B22 10X Buffer for Eco52I 1 ml 160 

B23 10X Buffer for SduI, Ppu21I 1 ml 160 

B24 10X Buffer for SdaI 1 ml 160 

B25 10X Buffer for Eam1105I 1 ml 160 

B26 10X Buffer for Ecl136II, PacI, SacI 1 ml 160 

B27 10X Buffer for AarI, AjiI, Bpu10I, ScaI, 

PasI 

1 ml 160 

B28 10X Buffer for TaqI 1 ml 160 

B29 10X Buffer for KpnI 1 ml 160 

B30 Buffer Set for Restriction Enzymes 5x1 ml 160 

B31 10X Buffer for BseXI 1 ml 160 

B43 10X Reaction Buffer with MgCl2 for 

DNase I, RNase-free 

1 ml 255 

B57 10X Buffer BamHI, Lsp1109I, SgeI 5x1 ml 160 

B59 10X Buffer BfuI 1 ml 160 

B69 10X T4 DNA Ligase Buffer 1.5 ml 239 

BB5 10X Buffer B 5x1 ml 160 

BG5 10X Buffer G 5x1 ml 160 

BO5 10X Buffer O 5x1 ml 160 

BR5 10X Buffer R 5x1 ml 160 

BY5 

DK 

10X Buffer Tango 5x1 ml 160 

DK0011 

EF 

Viral DNA/RNA Purification Kit (IVD) 50 preps 315 

EF0221 Pyrophosphatase, Inorganic (from 

yeast) 

10 u (0.1 u/μl) 272 

EF0651 FastAP Thermosensitive Alkaline 

Phosphatase 

1000 u (1 u/μl) 242 

EF0652 FastAP Thermosensitive Alkaline 

Phosphatase 

5x1000 u (1 u/μl) 242 

EF0654 

EK 


Phosphatase 

300 u (1 u/μl) 242 

EK0031 T4 Polynucleotide Kinase (T4 PNK) 500 u (10 u/μl) 243 

EK0032 

EL 

T4 Polynucleotide Kinase (T4 PNK) 2500 u (10 u/μl) 243 

EL0011 T4 DNA Ligase 1000 u (5 u/μl) 239 

EL0012 T4 DNA Ligase 5x1000 u (5 u/μl) 239 

EL0013 T4 DNA Ligase, HC 5000 u (30 u/μl) 239 

EL0014 T4 DNA Ligase 200 u (5 u/μl) 239 

EL0016 T4 DNA Ligase, LC 2x500 u (1 u/μl) 239 

EL0021 

EM 

T4 RNA Ligase 1000 u (10 u/μl) 240 

EM0821 

EN 

10X M.SssI Buffer, 50X (5 mM) SAM 25 μl 401 

EN0141 Endonuclease V, T.maritima (Endo V) 250 u (5 u/μl) 257 

EN0181 Micrococcal Nuclease 8000 u (300 u/μl) 268 

EN0191 Exonuclease III (Exo III) 4000 u (200 u/μl) 259 

EN0201 RNase H 100 u (5 u/μl) 266 

EN0202 RNase H 500 u (5 u/μl) 266 

EN0321 S1 Nuclease 10000 u (100 u/μl) 269 

EN0361 Uracil-DNA Glycosylase (UDG, UNG) 200 u (1 u/μl) 273 

EN0362 Uracil-DNA Glycosylase (UDG, UNG) 5x200 u (1 u/μl) 273 

EN0521 DNase I, RNase-free 1000 u (1 u/μl) 255 

EN0523 DNase I, RNase-free, HC 1000 u (50 u/μl) 255 

Cat. # Product name Size Page 

EN0525 DNase I, RNase-free (supplied 

with MnCl2) 

1000 u (1 u/μl) 255 

EN0531 RNase A, DNase and Protease-free 10 mg (10 mg/ml) 262 

EN0541 RNase T1 100000 u (1000 u/μl) 263 

EN0542 RNase T1 500000 u (1000 u/μl) 263 

EN0551 RNase A, RNase T1 1 ml 264 

EN0561 Lambda Exonuclease 1000 u (10 u/μl) 260 

EN0562 Lambda Exonuclease 5000 u (10 u/μl) 260 

EN0581 Exonuclease I (Exo I) 4000 u (20 u/μl) 258 

EN0582 Exonuclease I (Exo I) 20000 u (20 u/μl) 258 

EN0591 Endonuclease IV, E.coli (Endo IV) 100 u (2 u/μl) 256 

EN0601 RNase I 1000 u (10 u/μl) 265 

EN0602 

EO 

RNase I 5000 u (10 u/μl) 265 

EO0381 RiboLock RNase Inhibitor 2500 u (40 u/μl) 271 

EO0382 RiboLock RNase Inhibitor 4x2500 u (40 u/μl) 271 

EO0384 RiboLock RNase Inhibitor 24x2500 u (40 u/μl) 271 

EO0461 Agarase 100 u (0.5 u/μl) 319 

EO0491 Proteinase K (recombinant), PCR grade 1 ml (14-22 mg/ml) 270 

EO0492 Proteinase K (recombinant), PCR grade 5x1 ml (14-22 mg/ml) 270 

EO0861 

EP 

WELQut Protease 500 u (5 u/μl) 285 

EP0041 DNA Polymerase I 500 u (10 u/μl) 246 

EP0042 DNA Polymerase I 2500 u (10 u/μl) 246 

EP0051 Klenow Fragment 300 u (10 u/μl) 247 

EP0052 Klenow Fragment 1500 u (10 u/μl) 247 

EP0054 Klenow Fragment, LC 300 u (2 u/μl) 247 

EP0061 T4 DNA Polymerase 100 u (5 u/μl) 249 



EP0091 phi29 DNA Polymerase 250 u (10 u/μl) 245 



EP0101 T3 RNA Polymerase 2000 u (20 u/μl) 331 


EP0103 T3 RNA Polymerase, HC 10000 u (>100 u/μl) 331 


EP0112 T7 RNA Polymerase 5x5000 u (20 u/μl) 330 

EP0113 T7 RNA Polymerase, HC 25000 u (>100 u/μl) 330 

EP0131 SP6 RNA Polymerase 2000 u (20 u/μl) 331 

EP0133 SP6 RNA Polymerase, HC 5000 u (>100 u/μl) 331 

EP0161 Terminal Deoxynucleotidyl 


500 u (20 u/μl) 251 

EP0162 Terminal Deoxynucleotidyl 


2500 u (20 u/μl) 251 

EP0421 Klenow Fragment, exo- 300 u (5 u/μl) 248 

EP0422 Klenow Fragment, exo- 1500 u (5 u/μl) 248 

EP0691 

ER 

Bsm DNA Polymerase, Large Fragment 1600 u (8 u/μl) 253 

ER0011 AluI 600 u (10 u/μl) 85 

ER0012 AluI 3000 u (10 u/μl) 85 

ER0021 Alw21I (BsiHKAI) 500 u (10 u/μl) 86 

ER0031 Alw26I (BsmAI) 1000 u (10 u/μl) 86 

ER0041 Alw44I (ApaLI) 1000 u (10 u/μl) 86 

ER0051 BamHI 4000 u (10 u/μl) 88 

ER0052 BamHI 5x4000 u (10 u/μl) 88 

ER0053 BamHI, HC 20000 u (50 u/μl) 88 

ER0055 BamHI 10000 u (10 u/μl) 88 

ER0061 BcnI (NciI) 1000 u (10 u/μl) 89 

ER0071 BglI 2000 u (10 u/μl) 90 

ER0072 BglI 5x2000 u (10 u/μl) 90 

ER0081 BglII 500 u (10 u/μl) 91 


476 


ER0082 BglII 2500 u (10 u/μl) 91 

ER0091 Bpu1102I (BlpI) 200 u (10 u/μl) 93 

ER0092 Bpu1102I (BlpI) 1000 u (10 u/μl) 93 

ER0111 Bsp68I (NruI) 800 u (10 u/μl) 98 

ER0121 Bsp119I (BstBI) 2500 u (10 u/μl) 99 

ER0131 Bsp120I (PspOMI) 1500 u (10 u/μl) 99 

ER0141 Bsu15I (ClaI) 600 u (10 u/μl) 103 



ER0151 BsuRI (HaeIII) 3000 u (10 u/μl) 103 

ER0171 Cfr9I (XmaI) 300 u (10 u/μl) 105 

ER0172 Cfr9I (XmaI) 1500 u (10 u/μl) 105 

ER0181 Cfr10I (BsrFI) 200 u (10 u/μl) 105 

ER0191 Cfr13I (Sau96I) 1000 u (10 u/μl) 105 

ER0201 Cfr42I (SacII) 1200 u (10 u/μl) 106 

ER0202 Cfr42I (SacII) 5x1200 u (10 u/μl) 106 

ER0205 Cfr42I (SacII) 2000 u (10 u/μl) 106 

ER0211 Csp6I (CviQI) 1500 u (10 u/μl) 107 

ER0221 DraI 1500 u (10 u/μl) 108 

ER0223 DraI, HC 7500 u (50 u/μl) 108 

ER0231 Eam1104I (EarI) 300 u (10 u/μl) 108 

ER0232 Eam1104I (EarI) 1500 u (10 u/μl) 108 

ER0241 Eam1105I (AhdI) 1000 u (10 u/μl) 108 

ER0251 Ecl136II (EcoICRI) 1500 u (10 u/μl) 109 

ER0261 EcoO109I (DraII) 2000 u (10 u/μl) 115 

ER0271 EcoRI 5000 u (10 u/μl) 115 

ER0272 EcoRI 5x5000 u (10 u/μl) 115 

ER0273 EcoRI, HC 25000 u (50 u/μl) 115 

ER0275 EcoRI 10000 u (10 u/μl) 115 

ER0281 Eco24I (BanII) 1500 u (10 u/μl) 109 

ER0291 Eco31I (BsaI) 1000 u (10 u/μl) 110 

ER0292 Eco31I (BsaI) 5000 u (10 u/μl) 110 

ER0301 Eco32I (EcoRV) 2000 u (10 u/μl) 110 

ER0302 Eco32I (EcoRV) 5x2000 u (10 u/μl) 110 

ER0303 Eco32I (EcoRV), HC 10000 u (50 u/μl) 110 

ER0305 Eco32I (EcoRV) 4000 u (10 u/μl) 110 

ER0311 Eco47I (AvaII) 800 u (10 u/μl) 110 

ER0312 Eco47I (AvaII) 4000 u (10 u/μl) 110 

ER0321 Eco47III (AfeI) 200 u (10 u/μl) 111 

ER0322 Eco47III (AfeI) 1000 u (10 u/μl) 111 

ER0331 Eco52I (EagI) 500 u (10 u/μl) 111 

ER0332 Eco52I (EagI) 2500 u (10 u/μl) 111 

ER0341 Eco57I (AcuI) 200 u (5 u/μl) 112 

ER0342 Eco57I (AcuI) 1000 u (5 u/μl) 112 

ER0361 Eco72I (PmlI) 2000 u (10 u/μl) 112 

ER0371 Eco81I (Bsu36I) 500 u (10 u/μl) 113 

ER0372 Eco81I (Bsu36I) 2500 u (10 u/μl) 113 

ER0381 Eco88I (AvaI) 1000 u (10 u/μl) 113 

ER0391 Eco91I (BstEII) 1000 u (10 u/μl) 113 

ER0392 Eco91I (BstEII) 5000 u (10 u/μl) 113 

ER0401 Eco105I (SnaBI) 600 u (10 u/μl) 114 

ER0402 Eco105I (SnaBI) 3000 u (10 u/μl) 114 

ER0411 Eco130I (StyI) 2500 u (10 u/μl) 114 

ER0421 Eco147I (StuI) 1000 u (10 u/μl) 114 

ER0422 Eco147I (StuI) 5000 u (10 u/μl) 114 

ER0431 ScaI 1000 u (10 u/μl) 142 

ER0432 ScaI 5000 u (10 u/μl) 142 

ER0441 EheI (SfoI) 500 u (10 u/μl) 116 

ER0451 Esp3I (BsmBI) 200 u (10 u/μl) 117 

ER0452 Esp3I (BsmBI) 1000 u (10 u/μl) 117 

ER0461 GsuI (BpmI) 100 u (5 u/μl) 118 

ER0462 GsuI (BpmI) 500 u (5 u/μl) 118 

ER0471 Hin1I (BsaHI) 300 u (10 u/μl) 119 



ER0481 Hin6I (HinP1I) 2000 u (10 u/μl) 120 

ER0491 HincII (HindII) 500 u (10 u/μl) 120 

ER0492 HincII (HindII) 2500 u (10 u/μl) 120 

ER0501 HindIII 5000 u (10 u/μl) 121 

ER0502 HindIII 5x5000 u (10 u/μl) 121 

ER0503 HindIII, HC 25000 u (50 u/μl) 121 

ER0505 HindIII 10000 u (10 u/μl) 121 

ER0511 HpaII 1000 u (10 u/μl) 121 

ER0512 HpaII 5000 u (10 u/μl) 121 

ER0521 KpnI 4000 u (10 u/μl) 123 

ER0522 KpnI 5x4000 u (10 u/μl) 123 

ER0523 KpnI, HC 20000 u (50 u/μl) 123 

ER0531 Kpn2I (BspEI) 500 u (10 u/μl) 123 

ER0532 Kpn2I (BspEI) 2500 u (10 u/μl) 123 

ER0541 MspI (HpaII) 3000 u (10 u/μl) 128 

ER0542 MspI (HpaII) 5x3000 u (10 u/μl) 128 

ER0551 MvaI (BstNI) 2000 u (10 u/μl) 129 

ER0561 MluI 1000 u (10 u/μl) 127 

ER0562 MluI 5000 u (10 u/μl) 127 

ER0571 NcoI 500 u (10 u/μl) 130 

ER0572 NcoI 2500 u (10 u/μl) 130 

ER0575 NcoI 1000 u (10 u/μl) 130 

ER0581 NdeI 500 u (10 u/μl) 131 

ER0582 NdeI 2500 u (10 u/μl) 131 

ER0585 NdeI 4000 u (10 u/μl) 131 

ER0591 NotI 300 u (10 u/μl) 132 

ER0592 NotI 1500 u (10 u/μl) 132 

ER0593 NotI, HC 1500 u (50 u/μl) 132 

ER0595 NotI 500 u (10 u/μl) 132 

ER0601 PaeI (SphI) 500 u (10 u/μl) 133 

ER0602 PaeI (SphI) 2500 u (10 u/μl) 133 

ER0611 PstI 3000 u (10 u/μl) 138 

ER0612 PstI 5x3000 u (10 u/μl) 138 

ER0615 PstI 10000 u (10 u/μl) 138 

ER0621 PvuI 300 u (10 u/μl) 139 

ER0622 PvuI 1500 u (10 u/μl) 139 

ER0631 PvuII 2500 u (10 u/μl) 140 

ER0633 PvuII, HC 12500 u (50 u/μl) 140 

ER0635 PvuII 5000 u (10 u/μl) 140 

ER0641 SalI 1500 u (10 u/μl) 141 

ER0642 SalI 5x1500 u (10 u/μl) 141 

ER0645 SalI 2000 u (10 u/μl) 141 

ER0651 SduI (Bsp1286I) 500 u (10 u/μl) 143 

ER0661 SmaI 1200 u (10 u/μl) 146 

ER0662 SmaI 5x1200 u (10 u/μl) 146 

ER0663 SmaI, HC 6000 u (50 u/μl) 146 

ER0665 SmaI 2000 u (10 u/μl) 146 

ER0671 TaqI 3000 u (10 u/μl) 149 

ER0672 TaqI 5x3000 u (10 u/μl) 149 

ER0681 XbaI 1500 u (10 u/μl) 153 

ER0682 XbaI 5x1500 u (10 u/μl) 153 

ER0683 XbaI, HC 7500 u (50 u/μl) 153 

ER0685 XbaI 3000 u (10 u/μl) 153 

ER0691 XhoI 2000 u (10 u/μl) 154 

ER0692 XhoI 5x2000 u (10 u/μl) 154 

ER0693 XhoI, HC 10000 u (50 u/μl) 154 

ER0695 XhoI 5000 u (10 u/μl) 154 

ER0701 Bst1107I (BstZ17I) 500 u (10 u/μl) 102 

ER0711 Van91I (PflMI) 400 u (10 u/μl) 152 

ER0712 Van91I (PflMI) 2000 u (10 u/μl) 152 

ER0721 BclI 1000 u (10 u/μl) 89 

ER0722 BclI 5000 u (10 u/μl) 89 

ER0725 BclI 3000 u (10 u/μl) 89


ER0731 Mph1103I (NsiI) 1000 u (10 u/μl) 128 

ER0732 Mph1103I (NsiI) 5000 u (10 u/μl) 128 

ER0741 CpoI (RsrII) 200 u (10 u/μl) 106 

ER0742 CpoI (RsrII) 1000 u (10 u/μl) 106 

ER0751 MunI (MfeI) 300 u (10 u/μl) 129 

ER0752 MunI (MfeI) 1500 u (10 u/μl) 129 

ER0761 Psp5II (PpuMI) 500 u (10 u/μl) 138 

ER0771 SspI 500 u (10 u/μl) 148 

ER0772 SspI 2500 u (10 u/μl) 148 

ER0781 Bsp143I (Sau3AI) 300 u (10 u/μl) 99 

ER0782 Bsp143I (Sau3AI) 1500 u (10 u/μl) 99 

ER0801 HinfI 2000 u (10 u/μl) 121 

ER0802 HinfI 5x2000 u (10 u/μl) 121 

ER0803 HinfI, HC 10000 u (50 u/μl) 121 

ER0811 MboI 300 u (10 u/μl) 126 

ER0812 MboI 1500 u (10 u/μl) 126 

ER0821 MboII 300 u (5 u/μl) 126 

ER0822 MboII 1500 u (5 u/μl) 126 

ER0831 BspTI (AflII) 1000 u (10 u/μl) 101 

ER0851 Pfl23II (BsiWI) 300 u (3 u/μl) 136 

ER0871 BseGI (BtssCI) 500 u (10 u/μl) 94 

ER0872 BseGI (FokI) 2500 u (10 u/μl) 94 

ER0881 BseNI (BsrI) 1000 u (10 u/μl) 96 

ER0882 BseNI (BsrI) 5000 u (10 u/μl) 96 

ER0891 Bsh1285I (BsiEI) 600 u (10 u/μl) 97 

ER0901 Acc65I (Asp718I) 1000 u (10 u/μl) 83 

ER0902 Acc65I (KpnI) 5000 u (10 u/μl) 83 

ER0911 VspI (AseI) 1000 u (10 u/μl) 152 

ER0912 VspI (AseI) 5000 u (10 u/μl) 152 

ER0921 Bsh1236I (BstUI) 500 u (10 u/μl) 97 

ER0922 Bsh1236I (BstUI) 2500 u (10 u/μl) 97 

ER0931 Bsp1407I (BsrGI) 300 u (10 u/μl) 100 

ER0932 Bsp1407I (BsrGI) 1500 u (10 u/μl) 100 

ER0941 Psp1406I (AclI) 300 u (10 u/μl) 138 

ER0942 Psp1406I (AclI) 1500 u (10 u/μl) 138 

ER0961 Mva1269I (BsmI) 200 u (10 u/μl) 130 

ER0962 Mva1269I (BsmI) 1000 u (10 u/μl) 130 

ER0971 NheI 500 u (10 u/μl) 131 

ER0972 NheI 2500 u (10 u/μl) 131 

ER0975 NheI 1000 u (10 u/μl) 131 

ER0981 Tru1I (MseI) 300 u (10 u/μl) 151 

ER0982 Tru1I (MseI) 1500 u (10 u/μl) 151 

ER0983 Tru1I (MseI), HC 1500 u (50 u/μl) 151 

ER0991 AatII 300 u (10 u/μl) 82 

ER0992 AatII 1500 u (10 u/μl) 82 

ER1001 BshNI (BanI) 2000 u (10 u/μl) 97 

ER1011 BpiI (BbsI) 200 u (10 u/μl) 92 

ER1012 BpiI (BbsI) 1000 u (10 u/μl) 92 

ER1021 BstXI 500 u (10 u/μl) 102 

ER1022 BstXI 2500 u (10 u/μl) 102 

ER1031 KspAI (HpaI) 500 u (10 u/μl) 124 

ER1032 KspAI (HpaI) 2500 u (10 u/μl) 124 

ER1071 MnlI 300 u (10 u/μl) 127 

ER1072 MnlI 1500 u (10 u/μl) 127 

ER1081 BseDI (BsaJI) 300 u (10 u/μl) 94 

ER1082 BseDI (BsaJI) 1500 u (10 u/μl) 94 

ER1091 PauI (BssHII) 200 u (10 u/μl) 134 

ER1092 PauI (BssHII) 1000 u (10 u/μl) 134 

ER1101 HphI 300 u (10 u/μl) 122 

ER1102 HphI 1500 u (10 u/μl) 122 

ER1121 RsaI 1000 u (10 u/μl) 140 

ER1122 RsaI 5000 u (10 u/μl) 140 

ER1131 SacI 1200 u (10 u/μl) 141 


ER1132 SacI 5x1200 u (10 u/μl) 141 

ER1135 SacI 2000 u (10 u/μl) 141 

ER1141 TaiI (MaeII) 400 u (10 u/μl) 149 

ER1142 TaiI (MaeII) 2000 u (10 u/μl) 149 

ER1151 BspLI (NlaIV) 200 u (10 u/μl) 100 

ER1152 BspLI (NlaIV) 1000 u (10 u/μl) 100 

ER1161 BfmI (SfcI) 200 u (10 u/μl) 90 

ER1162 BfmI (SfcI) 1000 u (10 u/μl) 90 

ER1181 Bpu10I 200 u (5 u/μl) 93 

ER1191 SdaI (SbfI) 300 u (10 u/μl) 143 

ER1192 SdaI (SbfI) 1500 u (10 u/μl) 143 

ER1201 BseLI (BslI) 500 u (10 u/μl) 95 

ER1211 MlsI (MscI) 200 u (5 u/μl) 127 

ER1212 MlsI (MscI) 1000 u (5 u/μl) 127 

ER1221 NsbI (FspI) 400 u (10 u/μl) 132 

ER1231 AdeI (DraIII) 500 u (10 u/μl) 83 

ER1241 SmiI (SwaI) 1000 u (10 u/μl) 146 

ER1251 BcuI (SpeI) 400 u (10 u/μl) 89 

ER1252 BcuI (SpeI) 2000 u (10 u/μl) 89 

ER1261 BseMI (BsrDI) 100 u (5 u/μl) 95 

ER1262 BseMI (BsrDI) 500 u (5 u/μl) 95 

ER1271 MbiI (BsrBI) 1000 u (10 u/μl) 125 

ER1281 PagI (BspHI) 400 u (10 u/μl) 134 

ER1282 PagI (BspHI) 2000 u (10 u/μl) 134 

ER1291 TatI 100 u (5 u/μl) 150 

ER1301 XagI (EcoNI) 1000 u (10 u/μl) 152 

ER1311 BplI 100 u (5 u/μl) 92 

ER1312 BplI 500 u (5 u/μl) 92 

ER1321 BspPI (AlwI) 100 u (2 u/μl) 101 

ER1322 BspPI (AlwI) 500 u (2 u/μl) 101 

ER1331 PsyI (Tth111I) 1000 u (10 u/μl) 139 

ER1341 MssI (PmeI) 250 u (5 u/μl) 129 

ER1342 MssI (PmeI) 1250 u (5 u/μl) 129 

ER1351 TasI (Tsp509I) 1000 u (10 u/μl) 150 

ER1352 TasI (Tsp509I) 5000 u (10 u/μl) 150 

ER1361 TaaI (HpyCH4III) 200 u (10 u/μl) 149 

ER1362 TaaI (HpyCH4III) 1000 u (10 u/μl) 149 

ER1371 SchI (MlyI) 1000 u (10 u/μl) 143 

ER1381 XapI (ApoI) 500 u (10 u/μl) 153 

ER1391 CaiI (AlwNI) 500 u (10 u/μl) 104 

ER1401 BseMII (BspCNI) 100 u (1 u/μl) 95 

ER1411 ApaI 3000 u (10 u/μl) 87 

ER1415 ApaI 5000 u (10 u/μl) 87 

ER1421 Bme1390I (ScrFI) 500 u (10 u/μl) 91 

ER1422 Bme1390I (ScrFI) 2500 u (10 u/μl) 91 

ER1431 BoxI (PshAI) 500 u (10 u/μl) 92 

ER1441 BseSI (Bme1580I) 500 u (10 u/μl) 96 

ER1451 BseXI (BbvI) 100 u (3 u/μl) 96 

ER1452 BseXI (BbvI) 500 u (3 u/μl) 96 

ER1461 BshTI (AgeI) 200 u (10 u/μl) 98 

ER1462 BshTI (AgeI) 1000 u (10 u/μl) 98 

ER1471 XceI (NspI) 500 u (10 u/μl) 153 

ER1472 XceI (NspI) 2500 u (10 u/μl) 153 

ER1481 XmiI (AccI) 400 u (10 u/μl) 154 

ER1482 XmiI (AccI) 2000 u (10 u/μl) 154 

ER1491 AloI 100 u (2 u/μl) 85 

ER1501 BfuI (BciVI) 100 u (5 u/μl) 90 

ER1511 NmuCI (Tsp45I) 200 u (10 u/μl) 132 

ER1521 PdiI (NaeI) 200 u (10 u/μl) 135 

ER1522 PdiI (NaeI) 1000 u (10 u/μl) 135 

ER1531 PdmI (XmnI) 500 u (10 u/μl) 135 

ER1532 PdmI (XmnI) 2500 u (10 u/μl) 135 

ER1551 PsuI (BstYI) 500 u (5 u/μl) 139 


478 


ER1561 XmaJI (AvrII) 200 u (10 u/μl) 154 

ER1562 XmaJI (AvrII) 1000 u (10 u/μl) 154 

ER1571 Hpy8I (MjaIV) 200 u (10 u/μl) 122 

ER1572 Hpy8I (MjaIV) 1000 u (10 u/μl) 122 

ER1581 AarI 25 u (2 u/μl) 82 

ER1582 AarI 125 u (2 u/μl) 82 

ER1621 LweI (SfaNI) 100 u (10 u/μl) 125 

ER1622 LweI (SfaNI) 500 u (10 u/μl) 125 

ER1631 OliI (AleI) 200 u (10 u/μl) 133 

ER1632 OliI (AleI) 1000 u (10 u/μl) 133 

ER1641 SatI (Fnu4HI) 200 u (10 u/μl) 142 

ER1642 SatI (Fnu4HI) 1000 u (10 u/μl) 142 

ER1651 TauI 50 u (3 u/μl) 150 

ER1652 TauI 250 u (3 u/μl) 150 

ER1661 FspAI 100 u (5 u/μl) 118 

ER1662 FspAI 500 u (5 u/μl) 118 

ER1681 Nb.Bpu10I 1000 u (5 u/μl) 155 

ER1701 DpnI 500 u (10 u/μl) 107 

ER1702 DpnI 2500 u (10 u/μl) 107 

ER1705 DpnI 1000 u (10 u/μl) 107 

ER1711 BseJI (BsaBI) 2000 u (10 u/μl) 94 

ER1731 HpyF10VI (MwoI) 300 u (10 u/μl) 123 

ER1732 HpyF10VI (MwoI) 1500 u (10 u/μl) 123 

ER1741 BveI (BspMI) 250 u (5 u/μl) 104 

ER1751 PfoI 200 u (10 u/μl) 136 

ER1761 FspBI (BfaI) 500 u (10 u/μl) 118 

ER1762 FspBI (BfaI) 2500 u (10 u/μl) 118 

ER1771 I-SceI 250 u (10 u/μl) 157 

ER1781 PfeI (TfiI) 500 u (10 u/μl) 135 

ER1791 SsiI (AciI) 200 u (10 u/μl) 147 

ER1801 AlfI 50 u (2 u/μl) 85 

ER1811 FaqI (BsmFI) 100 u (2 u/μl) 117 

ER1821 SfiI 1000 u (10 u/μl) 144 

ER1831 Hin1II (NlaIII) 300 u (5 u/μl) 120 

ER1841 BauI (BssSI) 200 u (10 u/μl) 88 

ER1851 HhaI 2000 u (10 u/μl) 119 

ER1861 PasI 200 u (10 u/μl) 134 

ER1871 PscI (PciI) 200 u (10 u/μl) 137 

ER1872 PscI (PciI) 1000 u (10 u/μl) 137 

ER1881 HpyF3I (DdeI) 500 u (10 u/μl) 122 

ER1882 HpyF3I (DdeI) 2500 u (10 u/μl) 122 

ER1891 SgsI (AscI) 300 u (10 u/μl) 145 

ER1892 SgsI (AscI) 1500 u (10 u/μl) 145 

ER1901 CseI (HgaI) 100 u (5 u/μl) 106 

ER1921 EcoRII 200 u (10 u/μl) 116 

ER1931 LguI (SapI) 100 u (5 u/μl) 124 

ER1932 LguI (SapI) 500 u (5 u/μl) 124 

ER1941 AjiI (BmgBI) 200 u (5 u/μl) 84 

ER1951 AjuI 100 u (5 u/μl) 84 

ER1971 Ppu21I (BsaAI) 500 u (10 u/μl) 137 

ER1981 SmoI (SmlI) 200 u (10 u/μl) 147 

ER2001 RseI (MslI) 200 u (10 u/μl) 140 

ER2002 RseI (MslI) 1000 u (10 u/μl) 140 

ER2011 Nt.Bpu10I 1000 u (5 u/μl) 156 

ER2021 MreI (Sse232I) 300 u (10 u/μl) 128 

ER2031 SgrDI 200 u (5 u/μl) 145 

ER2041 BspOI (BmtI) 200 u (10 u/μl) 100 

ER2051 Nb.Mva1269I 1000 u (10 u/μl) 156 

ER2061 AanI (PsiI) 200 u (10 u/μl) 82 

ER2071 Lsp1109I (BbvI) 200 u (5 u/μl) 124 

ER2081 MauBI 100 u (5 u/μl) 125 

ER2091 SfaAI (AsiSI) 1000 u (10 u/μl) 144 

ER2101 TscAI (TspRI) 1000 u (10 u/μl) 151 



ER2191 SspDI (KasI) 250 u (10 u/μl) 148 

ER2201 PacI 250 u (10 u/μl) 133 

ER2202 PacI 1250 u (10 u/μl) 133 

ER2211 

F 

SgeI 250 u (3 u/μl) 145, 

402 

F-203S Terminal Transferase (TdT) 500 u (15 u/μl) 252 

F-203L Terminal Transferase (TdT) 2500 u (15 u/μl) 252 

F-541 Phusion Site-Directed Mutagenesis Kit 20 rxns incl. 

10 control rxns 

290 

F-602S PowerCut Dicer 60 u (1 u/μl) 267 

F-602L PowerCut Dicer 300 u (1 u/μl) 267 

F-610 Replicator RNAi Kit 40 rxns (of 50 μl) 329 

F-611S Phi6 RNA Replicase 60 u (1 u/μl) 332 

F-611L Phi6 RNA Replicase 300 u (1 u/μl) 332 

F-630 

FD 

Phi6 dsRNA 20 μg (0.5 μg/μl) 387 

FD0014 FastDigest AluI 100 rxns 16 

FD0024 FastDigest Alw21I 100 rxns 17 

FD0034 FastDigest Alw26I 100 rxns 17 

FD0044 FastDigest ApaLI (Alw44I) 200 rxns 18 

FD0054 FastDigest BamHI 800 rxns 20 

FD0055 FastDigest BamHI 2500 rxns 20 

FD0064 FastDigest NciI (BcnI) 200 rxns 51 

FD0074 FastDigest BglI 200 rxns 22 

FD0083 FastDigest BglII 100 rxns 23 

FD0084 FastDigest BglII 200 rxns 23 

FD0094 FastDigest BlpI (Bpu1102I) 50 rxns 23 

FD0124 FastDigest Bsp119I 200 rxns 29 


FD0143 FastDigest ClaI (Bsu15I) 50 rxns 34 

FD0144 FastDigest ClaI (Bsu15I) 100 rxns 34 

FD0154 FastDigest HaeIII (BsuRI) 400 rxns 42 

FD0184 FastDigest BsrFI (Cfr10I) 50 rxns 32 

FD0194 FastDigest Sau96I (Cfr13I) 100 rxns 62 

FD0214 FastDigest Csp6I 100 rxns 34 

FD0224 FastDigest DraI 200 rxns 35 

FD0234 FastDigest EarI (Eam1104I) 50 rxns 37 

FD0244 FastDigest Eam1105I 100 rxns 37 

FD0254 FastDigest Ecl136II 100 rxns 37 

FD0264 FastDigest EcoO109I 200 rxns 39 

FD0274 FastDigest EcoRI 800 rxns 39 

FD0275 FastDigest EcoRI 2500 rxns 39 

FD0293 FastDigest Eco31I 50 rxns 38 


FD0303 FastDigest EcoRV (Eco32I) 200 rxns 39 

FD0304 FastDigest EcoRV (Eco32I) 400 rxns 39 

FD0314 FastDigest AvaII (Eco47I) 200 rxns 20 

FD0324 FastDigest AfeI (Eco47III) 20 rxns 15 

FD0334 FastDigest EagI (Eco52I) 50 rxns 36 

FD0344 FastDigest AcuI (Eco57I) 20 rxns 14 

FD0364 FastDigest PmlI (Eco72I) 200 rxns 56 

FD0374 FastDigest Bsu36I (Eco81I) 50 rxns 34 

FD0384 FastDigest AvaI (Eco88I) 200 rxns 19 


FD0404 FastDigest SnaBI (Eco105I) 50 rxns 65 

FD0414 FastDigest StyI (Eco130I) 200 rxns 67 

FD0424 FastDigest StuI (Eco147I) 100 rxns 66 

FD0434 FastDigest ScaI 100 rxns 63 

FD0443 FastDigest EheI 20 rxns 40 

FD0444 FastDigest EheI 50 rxns 40 

FD0454 FastDigest BsmBI (Esp3I) 20 rxns 29 

FD0464 FastDigest BpmI (GsuI) 20 rxns 24


FD0474 FastDigest BsaHI (Hin1I) 100 rxns 26 

FD0484 FastDigest HinP1I (Hin6I) 200 rxns 44 

FD0494 FastDigest HincII 100 rxns 43 

FD0504 FastDigest HindIII 800 rxns 43 

FD0505 FastDigest HindIII 2500 rxns 43 

FD0514 FastDigest HpaII 200 rxns 44 

FD0524 FastDigest KpnI 300 rxns 45 

FD0534 FastDigest Kpn2I 100 rxns 46 

FD0544 FastDigest MspI 400 rxns 50 

FD0554 FastDigest MvaI 200 rxns 50 

FD0564 FastDigest MluI 100 rxns 47 

FD0573 FastDigest NcoI 20 rxns 52 



FD0583 FastDigest NdeI 100 rxns 52 



FD0593 FastDigest NotI 20 rxns 54 



FD0604 FastDigest SphI (PaeI) 20 rxns 66 

FD0614 FastDigest PstI 800 rxns 58 

FD0615 FastDigest PstI 2500 rxns 58 

FD0624 FastDigest PvuI 20 rxns 59 

FD0634 FastDigest PvuII 400 rxns 59 

FD0644 FastDigest SalI 200 rxns 61 

FD0654 FastDigest Bsp1286I (SduI) 50 rxns 30 

FD0663 FastDigest SmaI 100 rxns 65 

FD0664 FastDigest SmaI 200 rxns 65 

FD0674 FastDigest TaqI 400 rxns 68 

FD0684 FastDigest XbaI 300 rxns 71 

FD0685 FastDigest XbaI 750 rxns 71 

FD0694 FastDigest XhoI 400 rxns 71 

FD0695 FastDigest XhoI 1200 rxns 71 

FD0704 FastDigest BstZ17I (Bst1107I) 100 rxns 33 

FD0714 FastDigest PflMI (Van91I) 100 rxns 56 

FD0724 FastDigest BclI 300 rxns 22 

FD0734 FastDigest NsiI (Mph1103I) 100 rxns 54 

FD0744 FastDigest RsrII (CpoI) 20 rxns 60 

FD0753 FastDigest MfeI (MunI) 20 rxns 47 

FD0754 FastDigest MfeI (MunI) 50 rxns 47 

FD0764 FastDigest PpuMI (Psp5II) 50 rxns 57 

FD0774 FastDigest SspI 100 rxns 66 

FD0784 FastDigest Sau3AI (Bsp143I) 40 rxns 62 

FD0804 FastDigest HinfI 400 rxns 43 

FD0814 FastDigest MboI 50 rxns 46 

FD0824 FastDigest MboII 50 rxns 47 

FD0834 FastDigest AflII (BspTI) 150 rxns 15 

FD0854 FastDigest BsiWI (Pfl23II) 50 rxns 28 

FD0874 FastDigest BseGI 100 rxns 26 

FD0884 FastDigest BseNI 100 rxns 27 

FD0894 FastDigest BsiEI (Bsh1285I) 100 rxns 28 

FD0904 FastDigest Acc65I 100 rxns 13 

FD0914 FastDigest AseI (VspI) 200 rxns 19 

FD0924 FastDigest Bsh1236I 100 rxns 27 



FD0944 FastDigest AclI (Psp1406I) 20 rxns 14 

FD0964 FastDigest Mva1269I 50 rxns 51 

FD0973 FastDigest Nhe I 50 rxns 52 

FD0974 FastDigest NheI 100 rxns 52 

FD0984 FastDigest Tru1I 50 rxns 69 

FD0994 FastDigest AatII 50 rxns 13 


FD1004 FastDigest BanI (BshNI) 300 rxns 21 

FD1014 FastDigest BbsI (BpiI) 20 rxns 21 

FD1024 FastDigest BstXI 100 rxns 33 

FD1034 FastDigest HpaI (KspAI) 50 rxns 44 

FD1074 FastDigest MnlI 50 rxns 48 

FD1084 FastDigest BsaJI (BseDI) 20 rxns 26 

FD1124 FastDigest RsaI 100 rxns 60 

FD1133 FastDigest SacI 100 rxns 60 

FD1134 FastDigest SacI 200 rxns 60 

FD1144 FastDigest TaiI 50 rxns 68 

FD1154 FastDigest NlaIV (BspLI) 20 rxns 53 

FD1164 FastDigest SfcI (BfmI) 20 rxns 64 

FD1184 FastDigest Bpu10I 20 rxns 25 

FD1194 FastDigest SbfI (SdaI) 20 rxns 62 

FD1204 FastDigest BslI (BseLI) 100 rxns 28 

FD1214 FastDigest MscI (MlsI) 50 rxns 49 

FD1224 FastDigest FspI (NsbI) 50 rxns 41 

FD1234 FastDigest DraIII (AdeI) 50 rxns 36 

FD1244 FastDigest SwaI (SmiI) 200 rxns 67 

FD1253 FastDigest SpeI (BcuI) 20 rxns 65 

FD1254 FastDigest SpeI (BcuI) 50 rxns 65 

FD1264 FastDigest BsrDI (BseMI) 20 rxns 32 

FD1274 FastDigest BsrBI (MbiI) 100 rxns 32 

FD1284 FastDigest BspHI (PagI) 50 rxns 31 

FD1294 FastDigest TatI 20 rxns 68 

FD1304 FastDigest EcoNI (XagI) 100 rxns 38 

FD1314 FastDigest BplI 20 rxns 24 

FD1334 FastDigest PsyI 100 rxns 59 

FD1344 FastDigest MssI 100 rxns 50 

FD1354 FastDigest Tsp509I (TasI) 100 rxns 70 

FD1364 FastDigest TaaI 20 rxns 67 

FD1374 FastDigest MlyI (SchI) 100 rxns 48 

FD1383 FastDigest XapI 50 rxns 70 

FD1384 FastDigest XapI 100 rxns 70 

FD1394 FastDigest AlwNI (CaiI) 50 rxns 17 

FD1404 FastDigest BspCNI (BseMII) 20 rxns 31 

FD1414 FastDigest ApaI 300 rxns 18 

FD1424 FastDigest ScrFI (Bme1390I) 100 rxns 63 

FD1434 FastDigest PshAI (BoxI) 100 rxns 57 

FD1444 FastDigest Bme1580I (BseSI) 50 rxns 23 

FD1454 FastDigest BseXI 20 rxns 27 

FD1464 FastDigest AgeI (BshTI) 20 rxns 15 

FD1474 FastDigest NspI (XceI) 50 rxns 55 

FD1484 FastDigest AccI (XmiI) 50 rxns 13 

FD1514 FastDigest NmuCI 20 rxns 53 

FD1524 FastDigest NaeI (PdiI) 50 rxns 51 

FD1534 FastDigest PdmI 100 rxns 55 

FD1554 FastDigest PsuI 150 rxns 58 

FD1564 FastDigest AvrII (XmaJI) 20 rxns 20 

FD1574 FastDigest Hpy8I 20 rxns 45 

FD1634 FastDigest AleI (OliI) 20 rxns 16 

FD1644 FastDigest Fnu4HI (SatI) 20 rxns 40 

FD1654 FastDigest TauI 20 rxns 69 

FD1664 FastDigest FspAI 20 rxns 41 

FD1703 FastDigest DpnI 50 rxns 35 

FD1704 FastDigest DpnI 100 rxns 35 

FD1714 FastDigest BsaBI (BseJI) 200 rxns 25 

FD1724 FastDigest DrdI (AasI) 25 rxns 36 

FD1734 FastDigest HpyF10VI 20 rxns 45 

FD1744 FastDigest BspMI (BveI) 50 rxns 31 

FD1754 FastDigest PfoI 20 rxns 56 

FD1764 FastDigest BfaI (FspBI) 50 rxns 22 

FD1784 FastDigest TfiI (PfeI) 50 rxns 69 


480 


FD1794 FastDigest AciI (SsiI) 20 rxns 14 

FD1814 FastDigest BsmFI (FaqI) 20 rxns 29 

FD1824 FastDigest SfiI 150 rxns 64 

FD1834 FastDigest NlaIII (Hin1II) 100 rxns 53 

FD1854 FastDigest HhaI 200 rxns 42 

FD1884 FastDigest DdeI (HpyF3I) 50 rxns 35 

FD1894 FastDigest AscI (SgsI) 100 rxns 18 

FD1904 FastDigest HgaI (CseI) 20 rxns 42 

FD1934 FastDigest SapI (LguI) 20 rxns 61 

FD1954 FastDigest AjuI 20 rxns 16 

FD1974 FastDigest BsaAI (Ppu21I) 50 rxns 25 

FD2004 FastDigest MslI (RseI) 20 rxns 49 

FD2024 FastDigest MreI 20 rxns 48 

FD2044 FastDigest BmtI (BspOI) 20 rxns 24 

FD2064 FastDigest PsiI (AanI) 20 rxns 57 

FD2074 FastDigest BbvI (Lsp1109I) 100 rxns 21 

FD2084 FastDigest MauBI 20 rxns 46 

FD2094 FastDigest AsiSI (SfaAI) 100 rxns 19 

FD2104 FastDigest TspRI (TscAI) 100 rxns 70 

FD2114 FastDigest SexAI (CsiI) 50 rxns 63 

FD2124 FastDigest SfaNI (BmsI) 20 rxns 64 

FD2134 FastDigest BssHII (PteI) 20 rxns 33 

FD2144 FastDigest FokI 100 rxns 40 

FD2154 FastDigest NruI (RruI) 100 rxns 54 

FD2164 FastDigest SanDI (KflI) 20 rxns 61 

FD2174 FastDigest MseI (SaqAI) 50 rxns 49 

FD2184 FastDigest HaeII (BfoI) 200 rxns 41 

FD2204 FastDigest PacI 25 rxns 55 

FD2224 

K 

FastDigest PspFI 20 rxns 58 

K0441 TranscriptAid T7 High Yield 

Transcription Kit 

50 rxns 328 

K0481 GeneJET Plasmid Midiprep Kit 25 preps. 305 

K0482 GeneJET Plasmid Midiprep Kit 100 preps. 305 

K0491 GeneJET Plasmid Maxiprep Kit 10 preps. 306 

K0492 GeneJET Plasmid Maxiprep Kit 25 preps. 306 

K0502 GeneJET Plasmid Miniprep Kit 50 preps. 304 

K0503 GeneJET Plasmid Miniprep Kit 250 preps. 304 

K0512 Genomic DNA Purification Kit 100 preps. 311 

K0513 Silica Bead DNA Gel Extraction Kit 100 preps. (up to 

5 μg of DNA per prep) 

318 

K0621 DecaLabel DNA Labeling Kit 10 rxns 344 

K0622 DecaLabel DNA Labeling Kit 30 rxns 344 

K0651 Biotin DecaLabel DNA Labeling Kit 10 rxns 343 

K0652 Biotin DecaLabel DNA Labeling Kit 30 rxns 343 

K0661 Biotin Chromogenic Detection Kit 10 rxns 345 

K0662 Biotin Chromogenic Detection Kit 30 rxns 345 

K0691 GeneJET Gel Extraction Kit 50 preps. 317 

K0692 GeneJET Gel Extraction Kit 250 preps. 317 

K0701 GeneJET PCR Purification Kit 50 preps. 316 

K0702 GeneJET PCR Purification Kit 250 preps. 316 

K0721 GeneJET Genomic DNA Purification Kit 50 preps. 310 

K0722 GeneJET Genomic DNA Purification Kit 250 preps. 310 

K0731 GeneJET RNA Purification Kit 50 preps. 307 

K0732 GeneJET RNA Purification Kit 250 preps. 307 

K0761 GeneJET Whole Blood RNA 

Purification Mini Kit 

50 preps 308 

K0771 Fast DNA End Repair Kit 50 rxns 287 

K0781 GeneJET Whole Blood Genomic DNA 


50 preps 312 

K0782 GeneJET Whole Blood Genomic DNA 


250 preps 312 

K0791 GeneJET Plant Genomic DNA 


50 preps 313 



K0792 GeneJET Plant Genomic DNA 


250 preps 313 

K0801 GeneJET Plant RNA Purification Mini Kit 50 preps 309 

K0802 GeneJET Plant RNA Purification Mini Kit 250 preps 309 

K0821 GeneJET Viral DNA and RNA 

Purification Kit 

50 preps 314 

K1213 InsTAclone PCR Cloning Kit 10 rxns 10 rxns 281 

K1214 InsTAclone PCR Cloning Kit 30 rxns 30 rxns 281 

K1231 CloneJET PCR Cloning Kit 10 rxns 279 

K1232 CloneJET PCR Cloning Kit 40 rxns 279 

K1241 aLICator LIC Cloning 

and Expression Kit 1 

20 rxns 282 



20 rxns 282 



20 rxns 282 



30 rxns 282 


and Expression Set 1 

20 rxns 282 


and Expression Set 2 

30 rxns 282 

K1422 Rapid DNA Ligation Kit 50 rxns 329 

K1423 Rapid DNA Ligation Kit 150 rxns 329 

K1441 EpiJET DNA Methylation Analysis Kit 

(MspI/HpaII) 

200 rxns 399 

K1451 EpiJET DNA Methylation Analysis Kit 

(TaqI/HpyF30I) 

200 rxns 399 

K1481 EpiJET 5-hmC Analysis Kit 25 rxns 400 

K2561 Maxima H Minus Double-Stranded 

cDNA Synthesis Kit 

10 rxns 286 

K2710 TransformAid Bacterial 

Transformation Kit 

20 transf. 289 

K2711 

R 

TransformAid Bacterial 

Transformation Kit 

40 transf. 289 

R0391 IPTG, dioxane-free 1 g 424 

R0392 IPTG, dioxane-free 5 g 424 

R0393 IPTG, dioxane-free 5x5 g 424 

R0401 X-Gal 0.5 g 424 

R0402 X-Gal 2x1 g 424 

R0404 X-Gal 1 g 424 

R0421 dm4CTP, 10 mM 0.1 ml 415 

R0431 dm5CTP, 10 mM 0.1 ml 415 

R0441 ATP, 100 mM 0.25 ml 411 

R0451 CTP, 100 mM 0.25 ml 412 

R0461 GTP, 100 mM 0.25 ml 412 

R0471 UTP, 100 mM 0.25 ml 412 

R0481 NTP Set, 100 mM 4x0.25 ml 411 

R0491 TopVision Agarose 100 g 427 

R0492 TopVision Agarose 500 g 427 

R0501 dm6ATP, 10 mM Solution 0.1 ml 415 

R0551 Glycogen, RNA grade 2x0.1 ml (20 mg/ml) 423 

R0561 Glycogen, molecular biology grade 2x0.25 ml (20 mg/ml) 423 

R0581 Water, nuclease-free 4x1.25 ml 420 

R0582 Water, nuclease-free 30 ml 420 

R0601 DEPC-treated Water 30 ml 420 

R0603 DEPC-treated Water 5x1 ml 420 

R0641 2X RNA Loading Dye 1 ml 388 

R0801 TopVision Low Melting Point Agarose 25 g 427 

R0811 5-Fluoroorotic Acid 1 g 425 

R0812 5-Fluoroorotic Acid 5 g 425 

R0821 BCIP-T 1 g 426 

R0822 BCIP-T 5 g 426


R0841 NBT 1 g 426 

R0842 NBT 5 g 426 

R0851 X-Gluc 0.1 g 425 

R0852 X-Gluc 0.5 g 425 

R0861 DTT 5 g 420 

R0862 DTT 25 g 420 

R0941 X-Gal Solution, ready-to-use 10 ml (20 mg/ml) 424 

R0971 25 mM MgCl2 4x1.25 ml 421 

R1021 0.5 M EDTA, pH 8.0 5x1 ml 421 

R1091 Aminoallyl-UTP, 50 mM 0.05 ml 414 

R1101 Aminoallyl-dUTP, 50 mM 0.05 ml 414 

R1171 IPTG Solution, ready-to-use 10x1.5 ml 424 

R1181 3 M Sodium Acetate Solution, pH 5.2, 


5x1 ml 421 

R1191 

SD 

dITP, 100 mM Solution 0.25 ml 410 

SD0011 Lambda DNA 500 μg 291 

SD0021 Lambda DNA (dam-, dcm-) 500 μg 291 

SD0031 X174 RF1 DNA 50 μg 291 

SD0041 pBR322 DNA 100 μg 291 

SD0051 pUC18 DNA 50 μg 292 

SD0061 pUC19 DNA 50 μg 292 

SD0141 pTZ19R DNA 50 μg 292 

SD0171 pUC57 DNA 50 μg 292 

SD1111 Jurkat Genomic DNA 15 μg 403 

SD1121 CpG Methylated Jurkat Genomic DNA 15 μg 403 

SD1131 

SM 

CpG Methylated Human Genomic DNA 15 μg 403 

SM0101/ Lambda DNA/HindIII Marker, 2 

2/3 

5x50 μg 369 

SM0191/ Lambda DNA/EcoRI+HindIII Marker, 3 

2/3 

5x50 μg 369 

SM0221/ pUC19 DNA/MspI (HpaII) Marker, 23 

2/3 

50 μg 369 

SM0241/ GeneRuler 100 bp DNA Ladder 

2/3 

50 μg 360 

SM0251/ X174 DNA/BsuRI (HaeIII) Marker, 9 

2/3 

50 μg 369 

SM0271 pBR322 DNA/BsuRI (HaeIII) Marker, 5 50 μg 369 

SM0281 Lambda DNA/EcoRI Marker, 1 5x50 μg 369 

SM0311/ GeneRuler 1 kb DNA Ladder 

2/3 

5x50 μg 360 

SM0331/ GeneRuler DNA Ladder Mix 

2/3 

5x50 μg 360 

SM0371/ GeneRuler 50 bp DNA Ladder 

2/3 

50 μg 360 

SM0383 MassRuler Low Range DNA Ladder, 

ready-to-use 

2x500 μl 363 

SM0393 MassRuler High Range DNA Ladder, 

ready-to-use 

2x500 μl 363 

SM0403 MassRuler DNA Ladder Mix, readyto-use 

2x500 μl 363 

SM0613 O’RangeRuler 50 bp DNA Ladder, 

ready-to-use 

25 μg 367 


ready-to-use 

25 μg 367 


ready-to-use 

25 μg 367 


ready-to-use 

25 μg 367 

SM0653 O’RangeRuler 100 bp+500 bp DNA 


25 μg 367 

SM1103 FastRuler Low Range DNA Ladder, 

ready-to-use 

2x500 μl 364 

SM1113 FastRuler Middle Range DNA Ladder, 

ready-to-use 

2x500 μl 364 


SM1123 FastRuler High Range DNA Ladder, 

ready-to-use 

2x500 μl 364 

SM1133 O’GeneRuler 50 bp DNA Ladder, 

ready-to-use 

50 μg 360 

SM1143 O’GeneRuler 100 bp DNA Ladder, 

ready-to-use 

50 μg 360 

SM1153 O’GeneRuler 100 bp Plus DNA Ladder, 

ready-to-use 

50 μg 360 

SM1163 O’GeneRuler 1 kb DNA Ladder, 

ready-to-use 

5x50 μg 360 

SM1173 O’GeneRuler DNA Ladder Mix, 

ready-to-use 

5x50 μg 360 

SM1191/ GeneRuler Low Range DNA Ladder 

2/3 

50 μg 361 

SM1203 O’GeneRuler Low Range DNA Ladder, 

ready-to-use 

50 μg 363 

SM1211/ GeneRuler Ultra Low Range DNA 

50 μg 361 

2/3 Ladder 

SM1223 O’GeneRuler Ultra Low Range DNA 


50 μg 361 

SM1233 FastRuler Ultra Low Range DNA Ladder, 

ready-to-use 

2x500 μl 364 

SM1243 MassRuler Express HR Forward DNA 


2x500 μl 363 

SM1253 MassRuler Express HR Reverse DNA 


2x500 μl 363 

SM1263 MassRuler Express LR Forward DNA 


2x500 μl 363 

SM1273 MassRuler Express LR Reverse DNA 


2x500 μl 363 

SM1283 MassRuler Express Forward DNA 

Ladder Mix, ready-to-use 

2x500 μl 363 

SM1293 MassRuler Express Reverse DNA 

Ladder Mix, ready-to-use 

2x500 μl 363 


ready-to-use 

50 μg 367 


ready-to-use 

50 μg 367 


ready-to-use 

50 μg 367 

SM1343 O’GeneRuler 1 kb Plus DNA Ladder, 

ready-to-use 

5x50 μg 360 

SM1351/ GeneRuler High Range DNA Ladder 

2/3 

50 μg 361 

SM1551/ GeneRuler Express DNA Ladder 

2/3 

50 μg 361 

SM1563 O’GeneRuler Express DNA Ladder, 

ready-to-use 

50 μg 361 

SM1821 RiboRuler High Range RNA Ladder 5x20 μl 386 

SM1831 

SO 

RiboRuler Low Range RNA Ladder 5x20 μl 386 

SO131 Oligo(dT)18 Primer 30 μg (100 μM) 417 

SO132 Oligo(dT)18 Primer 60 μg (100 μM) 417 

SO142 Random Hexamer Primer 24 μg (100 μM) 417 

SO181 Exo-Resistant Random Primer 100 μl (500 μM) 417 


482 

Alphabetical Index 

A 

AanI ............................................................................................ 57, 82 

AarI ...................................................................................................82 

AasI ..................................................................................................36 

AatII ............................................................................................ 13, 82 

Abbreviations ...................................................................................471 

Acc65I ........................................................................................ 13, 83 

AccI .......................................................................................... 13, 154 

AciI ........................................................................................... 14, 147 

AclI ........................................................................................... 14, 138 

Activity Assay for 

Conventional Restriction Enzymes ...................................................77 

DNA/RNA Modifying Enzymes .......................................................236 

FastDigest Restriction Enzymes .........................................................8 

Activity of 

DNA/RNA modifying enzymes in Thermo Scientific buffers ..............274 

Mesophilic and Thermophilic Enzymes at 37°C ..............................165 

Restriction Enzymes in Five Buffer System .....................................161 

AcuI .......................................................................................... 14, 112 

AdeI ............................................................................................ 36, 83 

AfeI ........................................................................................... 15, 111 

AflII ........................................................................................... 15, 101 

Agarase...........................................................................................319 

Agarose...........................................................................................427 

AgeI ............................................................................................ 15, 98 

AhdI ................................................................................................108 

AjiI ....................................................................................................84 

AjuI ............................................................................................. 16, 84 

AleI ........................................................................................... 16, 133 

AlfI ....................................................................................................85 

aLICator Ligation Independent Cloning (LIC) 

and Expression System ....................................................................282 


FastAP Thermosensitive Alkaline Phosphatase ...............................242 

AloI ...................................................................................................85 

Alphabetic List of 

Commercially Available Restriction Enzymes ..................................205 

Conventional Restriction Enzymes ...................................................78 

Enzymes Cleaving DNA on the Both Sides of their Recognition 

Sequence ....................................................................................228 

Enzymes Generating 3’-protruding Ends ........................................226 


Enzymes Generating Blunt Ends ....................................................228 

FastDigest Restriction Enzymes .........................................................9 

Recognition Sequences ................................................................217 

AluI ............................................................................................. 16, 85 

Alw21I ........................................................................................ 17, 86 

Alw26I ........................................................................................ 17, 86 

Alw44I ........................................................................................ 18, 86 

AlwI ................................................................................................101 

AlwNI ........................................................................................ 17, 104 

Amino Acids 

Abbreviations and Structure ..........................................................463 

Codons ........................................................................................463 

Aminoallyl-dUTP ..............................................................................414 

Aminoallyl-UTP ................................................................................414 

Amplification ............................................. See in vitro transcription, PCR, 

Real-time PCR, Strand Displacement 

ApaI ............................................................................................ 18, 87 


ApaLI .......................................................................................... 18, 86 

ApoI ................................................................................................153 

AscI .......................................................................................... 18, 145 

AseI .......................................................................................... 19, 152 

AsiSI ......................................................................................... 19, 144 

Asp718I ............................................................................................83 

Assay 

Activity Assay ..................................................................... 8, 77, 236 

Contaminant Activities .................................................... 4, 8, 77, 236 

Double-stranded Endodeoxyribonuclease (dsENDO) ........................236 

E.coli Genomic DNA .....................................................................236 

Exodeoxyribonuclease ..................................................................236 

Functional....................................................................................236 

Labeled Oligonucleotide (LO)........................................... 7, 8, 77, 236 

Non-specific Nuclease and Cross-contamination ..............................77 

Protease ......................................................................................236 

Ribonuclease (RNase) ...................................................................236 

Single-stranded Endodeoxyribonuclease (ssENDO) .........................236 

ATP .................................................................................................411 

AvaI .......................................................................................... 19, 113 

AvaII .......................................................................................... 20, 110 

AvrII .......................................................................................... 20, 154 

B 

Bacterial Strain Genotypes ................................................................429 

Bacterial Transformation System ....................................... 286, 289, 290 

BamHI ......................................................................................... 20, 88 

BanI ............................................................................................ 21, 97 

BanII ...............................................................................................109 

BauI ..................................................................................................88 

BbsI ............................................................................................ 21, 92 

BbvI .................................................................................... 21, 96, 124 

BCIP-T ............................................................................................426 

BCIP-T/NBT Substrate Solution .........................................................426 

BciVI .................................................................................................90 

BclI ............................................................................................. 22, 89 

BcnI ............................................................................................ 51, 89 

BcuI ............................................................................................ 65, 89 

BfaI ........................................................................................... 22, 118 

BfmI ............................................................................................ 64, 90 

BfoI ...................................................................................................41 

BfuI ...................................................................................................90 

BglI ............................................................................................. 22, 90 

BglII ............................................................................................ 23, 91 

Biotin-11-dUTP ................................................................................414 

Biotin Chromogenic Detection Kit ......................................................345 

Biotin DecaLabel DNA Labeling Kit ....................................................343 

Blotting 

Southern .....................................................................................351 

BlpI ............................................................................................. 23, 93 

Blue/White (B/W) Cloning Assay ............................................................8 

Bme1390I ................................................................................... 63, 91 

Bme1580I ................................................................................... 23, 96 

BmgBI ...............................................................................................84 

BmsI .................................................................................................64 

BmtI .......................................................................................... 24, 100 

Bovine Serum Albumin (BSA) ............................................................422 

BoxI ............................................................................................ 57, 92 

BpiI ............................................................................................. 21, 92

BplI ............................................................................................. 24, 92 

BpmI ......................................................................................... 24, 118 

Bpu10I ........................................................................................ 25, 93 

Bpu1102I .................................................................................... 23, 93 

BsaAI ........................................................................................ 25, 137 

BsaBI .......................................................................................... 25, 94 

BsaHI ........................................................................................ 26, 119 

BsaI ................................................................................................110 

BsaJI ........................................................................................... 26, 94 

BseDI .......................................................................................... 26, 94 

BseGI .......................................................................................... 26, 94 

BseJI ........................................................................................... 25, 94 

BseLI........................................................................................... 28, 95 

BseMI ......................................................................................... 32, 95 

BseMII ......................................................................................... 31, 95 

BseNI .......................................................................................... 27, 96 

BseSI .......................................................................................... 23, 96 

BseXI .......................................................................................... 27, 96 

Bsh1236I .................................................................................... 27, 97 

Bsh1285I .................................................................................... 28, 97 

BshNI .......................................................................................... 21, 97 

BshTI .......................................................................................... 15, 98 

BsiEI ........................................................................................... 28, 97 

BsiHKAI .............................................................................................86 

BsiWI ........................................................................................ 28, 136 

BslI ............................................................................................. 28, 95 

BsmAI ...............................................................................................86 

BsmBI ....................................................................................... 29, 117 

Bsm DNA Polymerase, Large Fragment .............................................253 

BsmFI ....................................................................................... 29, 117 

BsmI ...............................................................................................130 

Bsp68I ..............................................................................................98 

Bsp119I ...................................................................................... 29, 99 

Bsp120I ...................................................................................... 30, 99 

Bsp143I ...................................................................................... 62, 99 

Bsp1286I .................................................................................. 30, 143 

Bsp1407I .................................................................................. 30, 100 

BspCNI ........................................................................................ 31, 95 

BspEI ..............................................................................................123 

BspHI ........................................................................................ 31, 134 

BspLI ........................................................................................ 53, 100 

BspMI ....................................................................................... 31, 104 

BspOI ........................................................................................ 24, 100 

BspPI ..............................................................................................101 

BspTI ........................................................................................ 15, 101 

BsrBI ......................................................................................... 32, 125 

BsrDI ........................................................................................... 32, 95 

BsrFI ......................................................................................... 32, 105 

BsrGI ...............................................................................................100 

BsrI ...................................................................................................96 

BssHII........................................................................................ 33, 134 

BssSI ................................................................................................88 

Bst1107I ................................................................................... 33, 102 

BstBI .................................................................................................99 

BstEII ..............................................................................................113 

BstNI ...............................................................................................129 

BstUI .................................................................................................97 

BstXI ......................................................................................... 33, 102 

BstYI ...............................................................................................139 

BstZ17I ..................................................................................... 33, 102 

Bsu15I ...................................................................................... 34, 103 

Bsu36I ...................................................................................... 34, 113 

BsuRI ........................................................................................ 42, 103 

BtsCI .................................................................................................94 

Buffers for DNA and RNA Electrophoresis 

10X TBE Buffer .................................................................... 371, 389 

50X TAE Buffer .................................................................... 371, 389 

Buffers for Restriction Enzymes 

10X Buffer AarI, AjiI, Bpu10I, ScaI, PasI ..........................................160 

10X Buffer B ................................................................................160 

10X Buffer BamHI, Lsp1109I, SgeI ................................................160 

10X Buffer BfuI ............................................................................160 

10X Buffer BseXI ..........................................................................160 

10X Buffer Bsp143I .....................................................................160 

10X Buffer Cfr9I ...........................................................................160 

10X Buffer Cfr10I .........................................................................160 

10X Buffer Eam1105I...................................................................160 

10X Buffer Ecl136II, PacI, SacI ......................................................160 

10X Buffer Eco52I ........................................................................160 

10X Buffer EcoRI ..........................................................................160 

10X Buffer G ................................................................................160 

10X Buffer KpnI ...........................................................................160 

10X Buffer O ...............................................................................160 

10X Buffer R ................................................................................160 

10X Buffer SdaI ...........................................................................160 

10X Buffer SduI, Ppu21I ...............................................................160 

10X Buffer Tango .....................................................................160 

10X Buffer TaqI ............................................................................160 

Buffer Set for Restriction Enzymes.................................................160 

Dilution Buffer for Restriction Enzymes ..........................................160 

BveI .......................................................................................... 31, 104 

C 

CaiI ........................................................................................... 17, 104 

Carriers for Precipitation of Nucleic Acids ..........................................423 

Cfr9I ...............................................................................................105 

Cfr10I ....................................................................................... 32, 105 

Cfr13I ....................................................................................... 62, 105 

Cfr42I .............................................................................................106 

Chromogenic Substrates 

BCIP-T ........................................................................................426 

NBT ............................................................................................426 

ClaI ........................................................................................... 34, 103 

Classification of Restriction Enzymes .................................................171 

Cleland’s reagent .............................................................................420 

CloneJET PCR Cloning Kit .................................................................279 

Cloning 

CloneJET PCR Cloning Kit .............................................................279 

InsTAclone PCR Cloning Kit ...........................................................281 

Phage and Plasmid DNA ...............................................................432 

Rapid DNA Ligation Kit .................................................................288 

TransformAid Bacterial Transformation Kit ...................... 286, 289, 290 

Codons and Assigned Amino Acids ....................................................463 

Commercially Available Restriction Enzymes, Alphabetic List ...............205 

Concentrations of Acids and Bases ...................................................464 

Constants, Physical 

General .......................................................................................465 

of the Nucleoside Triphosphates and Related Compounds ...............464 

Conventional Lambda DNA Markers ..................................................368 

Conventional Restriction Enzymes .......................................................77 

Conversions of 

Nucleic Acids ...............................................................................465 

Oligonucleotides ..........................................................................465 


484 

CpG Methylation ..............................................................................176 

CpG Methylated Human Genomic DNA...............................................403 

CpG Methylated Jurkat Genomic DNA................................................403 

CpG Methyltransferase (M.SssI)......................................................... 401 

CpoI .......................................................................................... 60, 106 

CseI .......................................................................................... 42, 106 

CsiI ...................................................................................................63 

Csp6I ........................................................................................ 34, 107 

CTP.................................................................................................412 

Custom AND OEM Manufacturing ......................................................... IX 

Custom DNA Ladders Service ...........................................................370 

CviQI ...............................................................................................107 

D 

Dam Methylation ..............................................................................174 

dATP ...............................................................................................408 

Dcm Methylation ..............................................................................175 

dCTP ...............................................................................................408 

DdeI .......................................................................................... 35, 122 

DecaLabel DNA Labeling Kit .............................................................344 

Decay Factors for Calculating the Amount of Radioactivity ..................466 

Denaturing Polyacrylamide Gel Electrophoresis ..................................375 

Deoxyribonucleoside Triphosphates ...................................................406 

DEPC-treated Water .........................................................................420 

Dephosphorylation 


Detection 

Biotin Chromogenic Detection Kit ..................................................345 

dGTP ...............................................................................................409 

Digestion of PCR Products ........................................................ 158, 169 

Dilution Buffer for Restriction Enzymes ..............................................160 

Dilution of restriction enzymes ..........................................................158 

dITP ................................................................................................410 

dm 4 CTP ...........................................................................................415 

dm 5 CTP ...........................................................................................415 

dm 6 ATP ...........................................................................................415 

DNA 

Digestion ............................................................................... 72, 158 

Extraction ............................................................................ 317, 318 

Labeling .............................................................................. 341, 376 

Nick-Translation (protocols) .....................................................350 

Random-primed Labeling .......................................................350 

Ladders and Markers ...................................................................354 

Ligase .........................................................................................239 

Ligation ............................................................................... 288, 294 

Loading Dyes ........................................................................372-373 

Maps ...................................................................................432-456 

Migration in Agarose and Polyacrylamide Gels ...............................462 

Number of Restriction Sites in DNA Molecules ...............................457 

Transformation .............................................................................289 

DNA Ends Generated by Restriction Enzyme Cleavage 



Enzymes Generating Blunt Ends ....................................................228 

DNA Extraction from gels 

Agarase .......................................................................................319 

GeneJET Gel Extraction Kit ............................................................317 

Silica Bead DNA Gel Extraction Kit .................................................318 

DNA Labeling 

Biotin DecaLabel DNA Labeling Kit ................................................343 

DecaLabel DNA Labeling Kit .........................................................344 


Modified Nucleotides ....................................................................413 

Protocols .....................................................................................349 

DNA Ladders and Markers 

Group Selection Guide ..................................................................354 

Loading Dyes ...............................................................................372 

Protocols for Labeling ...................................................................349 

Range Selection Guide .................................................................355 

Reagents for Electrophoresis ........................................................371 

Reference Guide ..........................................................................356 

DNA Loading Dye, 6X .......................................................................373 

DNA Loading Dye & SDS Solution, 6X ................................................373 

DNA Polymerase I ............................................................................246 

DNA Quantification on Gels ...............................................................374 

DNase I, RNase-free .........................................................................255 

dNTP/dUTP Mix................................................................................408 

dNTP Mixes .....................................................................................408 

dNTP Set .........................................................................................408 

Double Digestion 

DoubleDigest Engine ....................................................................158 

in Five Buffer System ...................................................................158 

in the Universal Tango Buffer ........................................................158 

of pUC19 Multiple Cloning Site......................................................170 

Setting up ....................................................................................158 

Using FastDigest Enzymes ..............................................................72 

DpnI .......................................................................................... 35, 107 

DraI ........................................................................................... 35, 108 

DraII ................................................................................................115 

DraIII ........................................................................................... 36, 83 

DrdI...................................................................................................36 

DTT .................................................................................................420 

dTTP ...............................................................................................409 

dUTP ...............................................................................................409 

E 

EagI .......................................................................................... 36, 111 

Eam1104I ................................................................................. 37, 108 

Eam1105I ................................................................................. 37, 108 

EarI ........................................................................................... 37, 108 

Ecl136II ..................................................................................... 37, 109 

Eco24I ............................................................................................109 

Eco31I ...................................................................................... 38, 110 

Eco32I ...................................................................................... 39, 110 

Eco47I ...................................................................................... 20, 110 

Eco47III ..................................................................................... 15, 111 

Eco52I ...................................................................................... 36, 111 

Eco57I ...................................................................................... 14, 112 

Eco72I ...................................................................................... 56, 112 

Eco81I ...................................................................................... 34, 113 

Eco88I ...................................................................................... 19, 113 

Eco91I ...................................................................................... 38, 113 

Eco105I65, 114 

Eco130I .................................................................................... 67, 114 

Eco147I .................................................................................... 66, 114 

EcoBI Methylation ............................................................................179 

EcoICRI ...........................................................................................109 

EcoKI Methylation ............................................................................179 

E.coli Host Restriction and Modification Systems ................................431 

EcoNI ........................................................................................ 38, 152 

EcoO109I .................................................................................. 39, 115 

EcoRI ........................................................................................ 39, 115 

EcoRII ..............................................................................................116

EcoRV ....................................................................................... 39, 110 

EDTA, 0.5 M, pH 8.0 ........................................................................421 

EheI .......................................................................................... 40, 116 


Buffers ................................................................................ 371, 389 

DNA Electrophoresis .....................................................................353 

Loading Dyes ....................................................................... 372, 388 

RNA Electrophoresis .....................................................................385 

Endonuclease IV, E.coli (Endo IV) .......................................................256 

Endonuclease V, T.maritima (Endo V)..................................................257 

EpiJET DNA Methylation Analysis Kit (TaqI/HpyF30I)............................399 

EpiJET DNA Methylation Analysis Kit (MspI/HpaII)................................399 

EpiJET 5-hmC Analysis Kit................................................................400 

Esp3I ........................................................................................ 29, 117 

Estimation of pmol DNA Ends ...........................................................465 

Exodeoxyribonuclease Assay .............................................................236 

Exonuclease I (Exo I) .........................................................................258 

Exonuclease III (Exo III) ......................................................................259 

Exo-Resistant Random Primer ...........................................................417 

Express DNA Ladders ....................................................... 358, 362, 365 

F 

5-Fluoroorotic Acid ...........................................................................425 

FaqI .......................................................................................... 29, 117 

FastAP Thermosensitive Alkaline Phosphatase ...................................242 

FastDigest AatII ..................................................................................13 

FastDigest Acc65I ..............................................................................13 

FastDigest AccI (XmiI) .........................................................................13 

FastDigest AciI (SsiI) ...........................................................................14 

FastDigest AclI (Psp1406I) ..................................................................14 

FastDigest AcuI (Eco57I) .....................................................................14 

FastDigest AfeI (Eco47III) ....................................................................15 

FastDigest AflII (BspTI) ........................................................................15 

FastDigest AgeI (BshTI) .......................................................................15 

FastDigest AjuI ...................................................................................16 

FastDigest AleI (OliI) ...........................................................................16 

FastDigest AluI ...................................................................................16 

FastDigest Alw21I ..............................................................................17 

FastDigest Alw26I ..............................................................................17 

FastDigest AlwNI (CaiI) ........................................................................17 

FastDigest ApaI ..................................................................................18 

FastDigest ApaLI (Alw44I) ...................................................................18 

FastDigest AscI (SgsI) .........................................................................18 

FastDigest AseI (VspI) .........................................................................19 

FastDigest AsiSI (SfaAI) .......................................................................19 

FastDigest AvaI (Eco88I) .....................................................................19 

FastDigest AvaII (Eco47I) ....................................................................20 

FastDigest AvrII (XmaJI) ......................................................................20 

FastDigest BamHI ..............................................................................20 

FastDigest BanI (BshNI) ......................................................................21 

FastDigest BbsI (BpiI) .........................................................................21 

FastDigest BbvI (Lsp1109I) .................................................................21 

FastDigest BclI ...................................................................................22 

FastDigest BfaI (FspBI)........................................................................22 

FastDigest BglI ...................................................................................22 

FastDigest BglII ..................................................................................23 

FastDigest BlpI (Bpu1102I) .................................................................23 

FastDigest Bme1580I (BseSI)..............................................................23 

FastDigest BmtI (BspOI) ......................................................................24 

FastDigest BplI ...................................................................................24 

FastDigest BpmI (GsuI) .......................................................................24 

FastDigest Bpu10I ..............................................................................25 

FastDigest BsaAI (Ppu21I) ..................................................................25 

FastDigest BsaBI (BseJI) .....................................................................25 

FastDigest BsaHI (Hin1I) .....................................................................26 

FastDigest BsaJI (BseDI) .....................................................................26 

FastDigest BseGI ................................................................................26 

FastDigest BseNI ................................................................................27 

FastDigest BseXI ................................................................................27 

FastDigest Bsh1236I ..........................................................................27 

FastDigest BsiEI (Bsh1285I) ................................................................28 

FastDigest BsiWI (Pfl23II) ....................................................................28 

FastDigest BslI (BseLI) ........................................................................28 

FastDigest BsmBI (Esp3I) ....................................................................29 

FastDigest BsmFI (FaqI) ......................................................................29 

FastDigest Bsp119I ............................................................................29 

FastDigest Bsp120I ............................................................................30 

FastDigest Bsp1286I (SduI) ................................................................30 

FastDigest Bsp1407I ..........................................................................30 

FastDigest BspCNI (BseMII) .................................................................31 

FastDigest BspHI (PagI) ......................................................................31 

FastDigest BspMI (BveI) ......................................................................31 

FastDigest BsrBI (MbiI) .......................................................................32 

FastDigest BsrDI (BseMI) ....................................................................32 

FastDigest BsrFI (Cfr10I) .....................................................................32 

FastDigest BssHII (PteI) .......................................................................33 

FastDigest BstXI .................................................................................33 

FastDigest BstZ17I (Bst1107I) ............................................................33 

FastDigest Bsu36I (Eco81I) .................................................................34 

FastDigest ClaI (Bsu15I) .....................................................................34 

FastDigest Csp6I ................................................................................34 

FastDigest DdeI (HpyF3I).....................................................................35 

FastDigest DpnI .................................................................................35 

FastDigest DraI ..................................................................................35 

FastDigest DraIII (AdeI) .......................................................................36 

FastDigest DrdI (AasI) .........................................................................36 

FastDigest EagI (Eco52I) .....................................................................36 

FastDigest Eam1105I .........................................................................37 

FastDigest EarI (Eam1104I) ................................................................37 

FastDigest Ecl136II ............................................................................37 

FastDigest Eco31I ..............................................................................38 

FastDigest Eco91I ..............................................................................38 

FastDigest EcoNI (XagI) .......................................................................38 

FastDigest EcoO109I ..........................................................................39 

FastDigest EcoRI ................................................................................39 

FastDigest EcoRV (Eco32I) ..................................................................39 

FastDigest EheI ..................................................................................40 

FastDigest Fnu4HI (SatI) .....................................................................40 

FastDigest FokI ..................................................................................40 

FastDigest FspAI ................................................................................41 

FastDigest FspI (NsbI) .........................................................................41 

FastDigest HaeII (BfoI) ........................................................................41 

FastDigest HaeIII (BsuRI) .....................................................................42 

FastDigest HgaI (CseI) ........................................................................42 

FastDigest HhaI ..................................................................................42 

FastDigest HincII ................................................................................43 

FastDigest HindIII ...............................................................................43 

FastDigest HinfI ..................................................................................43 

FastDigest HinP1I (Hin6I) ....................................................................44 

FastDigest HpaII .................................................................................44 

FastDigest HpaI (KspAI) ......................................................................44 

FastDigest Hpy8I ................................................................................45 

FastDigest HpyF10VI ..........................................................................45 


486 

FastDigest Kpn2I ................................................................................46 

FastDigest KpnI ..................................................................................45 

FastDigest MauBI ...............................................................................46 

FastDigest MboI .................................................................................46 

FastDigest MboII ................................................................................47 

FastDigest MfeI (MunI)........................................................................47 

FastDigest MluI ..................................................................................47 

FastDigest MlyI (SchI) .........................................................................48 

FastDigest MnlI ..................................................................................48 

FastDigest MreI ..................................................................................48 

FastDigest MscI (MlsI) ........................................................................49 

FastDigest MseI (SaqAI) ......................................................................49 

FastDigest MslI (RseI) .........................................................................49 

FastDigest MspI .................................................................................50 

FastDigest MssI .................................................................................50 

FastDigest Mva1269I .........................................................................51 

FastDigest MvaI .................................................................................50 

FastDigest NaeI (PdiI) .........................................................................51 

FastDigest NciI (BcnI) .........................................................................51 

FastDigest NcoI ..................................................................................52 

FastDigest NdeI .................................................................................52 

FastDigest NheI .................................................................................52 

FastDigest NlaIII (Hin1II) ......................................................................53 

FastDigest NlaIV (BspLI) ......................................................................53 

FastDigest NmuCI ..............................................................................53 

FastDigest NotI ..................................................................................54 

FastDigest NruI (RruI) .........................................................................54 

FastDigest NsiI (Mph1103I) .................................................................54 

FastDigest NspI (XceI) .........................................................................55 

FastDigest PacI ..................................................................................55 

FastDigest PdmI .................................................................................55 

FastDigest PflMI (Van91I) ....................................................................56 

FastDigest PfoI ...................................................................................56 

FastDigest PmlI (Eco72I) .....................................................................56 

FastDigest PpuMI (Psp5II) ...................................................................57 

FastDigest PshAI (BoxI) .......................................................................57 

FastDigest PsiI (AanI) ..........................................................................57 

FastDigest PspFI ................................................................................58 

FastDigest PstI ...................................................................................58 

FastDigest PsuI ..................................................................................58 

FastDigest PsyI ..................................................................................59 

FastDigest PvuI ..................................................................................59 

FastDigest PvuII .................................................................................59 

FastDigest Restriction Enzymes 

Activity and Quality Control Assays ....................................................8 

Navigation Guide ..............................................................................5 

Product List .....................................................................................9 

Protocols and Recommendations ....................................................72 

Reaction Conditions .......................................................................73 

Storage and Shipping .......................................................................8 

FastDigest® RsaI...............................................................................60 

FastDigest RsrII (CpoI) ........................................................................60 

FastDigest SacI ..................................................................................60 

FastDigest SalI ...................................................................................61 

FastDigest SanDI (KflI) ........................................................................61 

FastDigest SapI (LguI) .........................................................................61 

FastDigest Sau3AI (Bsp143I) ..............................................................62 

FastDigest Sau96I (Cfr13I) ..................................................................62 

FastDigest SbfI (SdaI) .........................................................................62 

FastDigest ScaI ..................................................................................63 

FastDigest ScrFI (Bme1390I) ..............................................................63 

FastDigest SexAI (CsiI) ........................................................................63 


FastDigest SfaNI (BmsI) ......................................................................64 

FastDigest SfcI (BfmI) .........................................................................64 

FastDigest SfiI ....................................................................................64 

FastDigest SmaI .................................................................................65 

FastDigest SnaBI (Eco105I) .................................................................65 

FastDigest SpeI (BcuI) ........................................................................65 

FastDigest SphI (PaeI) ........................................................................66 

FastDigest SspI ..................................................................................66 

FastDigest StuI (Eco147I) ....................................................................66 

FastDigest StyI (Eco130I) ....................................................................67 

FastDigest SwaI (SmiI) ........................................................................67 

FastDigest TaaI ..................................................................................67 

FastDigest TaiI ...................................................................................68 

FastDigest TaqI ..................................................................................68 

FastDigest TatI ...................................................................................68 

FastDigest TauI ..................................................................................69 

FastDigest TfiI (PfeI) ...........................................................................69 

FastDigest Tru1I .................................................................................69 

FastDigest Tsp509I (TasI) ....................................................................70 

FastDigest TspRI (TscAI) .....................................................................70 

FastDigest XapI ..................................................................................70 

FastDigest XbaI ..................................................................................71 

FastDigest XhoI ..................................................................................71 

Fast DNA End Repair Kit ...................................................................287 

FastRuler High Range DNA Ladder, ready-to-use ................................364 

FastRuler Low Range DNA Ladder, ready-to-use ................................364 

FastRuler Middle Range DNA Ladder, ready-to-use ............................364 

FastRuler Ultra Low Range DNA Ladder, ready-to-use ........................364 

First Strand cDNA Synthesis 

RiboLock RNase Inhibitor ..............................................................271 

RNase H ......................................................................................266 

Fluorescein-12-dUTP .......................................................................413 

Fnu4HI ...................................................................................... 40, 142 

FokI...................................................................................................40 

FspAI ........................................................................................ 41, 118 

FspBI ........................................................................................ 22, 118 

FspI........................................................................................... 41, 132 

Functional Assay ..............................................................................236 

FX174 DNA /BsuRI (HaeIII) Marker, 9 .................................................368 

FX174 DNA /BsuRI (HaeIII) Marker, 9, ready-to-use ............................368 

FX174 DNA Restriction Map and Restriction Sites .......................435-436 

FX174 RF1 DNA ..............................................................................291 

G 

Gels 

Agarose ............................................................................... 374, 390 

Polyacrylamide .................................................................... 375, 391 

Gene Expression ..............................................................................512 

GeneJET Plant RNA Purification Mini Kit ............................................309 

GeneJET Plasmid Maxiprep Kit..........................................................306 

GeneJET Plasmid Midiprep Kit ..........................................................305 

GeneJET Gel Extraction Kit................................................................317 

GeneJET Genomic DNA Purification Kit ..............................................310 

GeneJET PCR Purification Kit ............................................................316 

GeneJET Plant Genomic DNA Purification Mini Kit...............................313 

GeneJET Plasmid Maxiprep Kit..........................................................306 

GeneJET Plasmid Midiprep Kit ..........................................................305 

GeneJET Plasmid Miniprep Kit ..........................................................304 

GeneJET RNA Purification Kit ............................................................307 

GeneJET Viral DNA and RNA Purification Kit .......................................314 

GeneJET Whole Blood Genomic DNA Purification Mini Kit ...................312

GeneJET Whole Blood RNA Purification Mini Kit .................................308 

Generated Recognition Sequences Resulting from 

Fill-in of 5’-overhang and Self-ligation ...........................................200 

Ligation of Blunt DNA Ends ...........................................................180 

Ligation of Protruding Compatible DNA Ends ..................................190 

Removal of 3’-overhang and Self-ligation ......................................204 

GeneRuler 1 kb DNA Ladder .............................................................359 

GeneRuler 1 kb DNA Ladder, ready-to-use .........................................359 

GeneRuler 1 kb Plus DNA Ladder ......................................................359 

GeneRuler 1 kb Plus DNA Ladder, ready-to-use .................................359 

GeneRuler 50 bp DNA Ladder ...........................................................359 

GeneRuler 50 bp DNA Ladder, ready-to-use ......................................359 

GeneRuler 100 bp DNA Ladder .........................................................359 

GeneRuler 100 bp DNA Ladder, ready-to-use ....................................359 

GeneRuler 100 bp Plus DNA Ladder ..................................................359 

GeneRuler100 bp Plus DNA Ladder, ready-to-use ..........................359 

GeneRuler DNA Ladder Mix ..............................................................359 

GeneRuler DNA Ladder Mix, ready-to-use ..........................................359 

GeneRuler Express DNA Ladder ........................................................359 

GeneRuler Express DNA Ladder, ready-to-use ....................................359 

GeneRuler High Range DNA Ladder ...................................................359 

GeneRuler High Range DNA Ladder, ready-to-use ..............................359 

GeneRuler Low Range DNA Ladder ...................................................359 

GeneRuler Low Range DNA Ladder, ready-to-use ...............................359 

GeneRuler Ultra Low Range DNA Ladder ...........................................359 

GeneRuler Ultra Low Range DNA Ladder, ready-to-use .......................359 

Genetic Markers ..............................................................................430 

Genome Sizes ..................................................................................461 

Genomic DNA Purification 

GeneJET Genomic DNA Purification Kit ..........................................310 

Genomic DNA Purification Kit ........................................................311 

Genomic DNA Purification Kit ............................................................311 

Genotypes .......................................................................................429 

Glycogen .........................................................................................423 

Glycosylase (UDG, UNG) ....................................................................273 

GsuI .......................................................................................... 24, 118 

GTP ................................................................................................412 

Guide for 

DNA Ladders and Markers (group selection guide) .........................354 

Enzymes & Kits for Molecular Cloning ............................................278 

Enzymes & Kits for Molecular Labeling & Detection ........................342 

In vitro Transcription .....................................................................326 

Navigation .......................................................................................5 

Nucleic Acid Purification ...............................................................303 

RNA Ladders ...............................................................................386 

Selection of Deoxyribonucleases (DNases) .....................................254 

Selection of Ribonucleases (RNases) .............................................261 

Selection of Sugar Nonspecific Nucleases ......................................268 

H 

HaeII .................................................................................................41 

HaeIII......................................................................................... 42, 103 

HgaI .......................................................................................... 42, 106 

HhaI .......................................................................................... 42, 119 

Hin1I ......................................................................................... 26, 119 

Hin1II ........................................................................................ 53, 120 

Hin6I ......................................................................................... 44, 120 

HincII......................................................................................... 43, 120 

HindII ..............................................................................................120 

HindIII ........................................................................................ 43, 121 

HinfI .......................................................................................... 43, 121 

HinP1I ....................................................................................... 44, 120 

Histochemical Detection of GUS ........................................................425 

Homing Enzyme ...............................................................................157 

HpaI .......................................................................................... 44, 124 

HpaII ..................................................................................44, 121, 128 

HphI ................................................................................................122 

Hpy8I ........................................................................................ 45, 122 

HpyCH4III ........................................................................................149 

HpyF3I ...................................................................................... 35, 122 

HpyF10VI................................................................................... 45, 123 

Hybridization 



I 

Icons, Description ........................................................................ 7, 237 

InsTAclone PCR Cloning Kit ...............................................................281 

IPTG ................................................................................................424 

IPTG Solution ...................................................................................424 

I-SceI ..............................................................................................157 

Isopropyl--D-thiogalactopyranoside (IPTG) .......................................424 

Isoschizomers 

Alphabetic List of Commercially Available 

Restriction Enzymes .....................................................................205 

J 

Jurkat Genomic DNA ........................................................................403 

K 

KasI ................................................................................................148 

KflI ....................................................................................................61 

Kinase .............................................................................................243 

Kits 

Biotin Chromogenic Detection Kit ..................................................345 






GeneJET PCR Purification Kit ........................................................316 

GeneJET Plasmid Maxiprep Kit ......................................................306 

GeneJET Plasmid Midiprep Kit ......................................................305 

GeneJET Plasmid Miniprep Kit ......................................................304 

GeneJET RNA Purification Kit ........................................................307 





TranscriptAid T7 High Yield Transcription Kit ...................................328 

TransformAid Bacterial Transformation Kit .......................286, 289, 290 

Klenow Fragment .............................................................................247 

Klenow Fragment, exo – .....................................................................248 

Kpn2I ........................................................................................ 46, 123 

KpnI .......................................................................................... 45, 123 

KspAI ........................................................................................ 44, 124 

L 

Labeled Oligonucleotide (LO) Test .........................................4, 8, 77, 236 

Labeling .................................................................................. 341, 376 


488 

Labeling Kits 



Ladders 

DNA .....................................................................................370-380 

FastRuler High Range DNA Ladder, ready-to-use ......................364 

FastRuler Low Range DNA Ladder, ready-to-use ......................364 

FastRuler Middle Range DNA Ladder, ready-to-use...................364 

FastRuler Ultra Low Range DNA Ladder, ready-to-use ...............364 

GeneRuler 1 kb DNA Ladder ...................................................359 

GeneRuler 1 kb DNA Ladder, ready-to-use ...............................359 

GeneRuler 1 kb Plus DNA Ladder ............................................359 

GeneRuler 1 kb Plus DNA Ladder, ready-to-use .......................359 

GeneRuler 50 bp DNA Ladder .................................................359 

GeneRuler 50 bp DNA Ladder, ready-to-use .............................359 

GeneRuler 100 bp DNA Ladder ...............................................359 

GeneRuler 100 bp DNA Ladder, ready-to-use ...........................359 

GeneRuler 100 bp Plus DNA Ladder ........................................359 

GeneRuler100 bp Plus DNA Ladder, ready-to-use ................359 

GeneRuler DNA Ladder Mix.....................................................359 

GeneRuler DNA Ladder Mix, ready-to-use ................................359 

GeneRuler Express DNA Ladder ..............................................359 

GeneRuler Express DNA Ladder, ready-to-use ..........................359 

GeneRuler High Range DNA Ladder .........................................359 

GeneRuler High Range DNA Ladder, ready-to-use ....................359 

GeneRuler Low Range DNA Ladder .........................................359 

GeneRuler Low Range DNA Ladder, ready-to-use .....................359 

GeneRuler Ultra Low Range DNA Ladder ..................................359 

GeneRuler Ultra Low Range DNA Ladder, ready-to-use .............359 

MassRuler DNA Ladder Mix, ready-to-use................................362 

MassRuler Express Forward DNA Ladder Mix, ready-to-use ......362 

MassRuler Express HR Forward DNA Ladder, ready-to-use .......362 

MassRuler Express HR Reverse DNA Ladder, ready-to-use ........362 

MassRuler Express LR Forward DNA Ladder, ready-to-use ........362 

MassRuler Express LR Reverse DNA Ladder, ready-to-use ........362 

MassRuler Express Reverse DNA Ladder Mix, ready-to-use ......362 

MassRuler High Range DNA Ladder, ready-to-use ....................362 

MassRuler Low Range DNA Ladder, ready-to-use .....................362 

O’GeneRuler 1 kb DNA Ladder, ready-to-use ...........................359 

O’GeneRuler 1 kb Plus DNA Ladder, ready-to-use ....................359 

O’GeneRuler 50 bp DNA Ladder, ready-to-use .........................359 

O’GeneRuler 100 bp DNA Ladder, ready-to-use .......................359 

O’GeneRuler 100 bp Plus DNA, ready-to-use ...........................359 

O’GeneRuler DNA Ladder Mix, ready-to-use ............................359 

O’GeneRuler Express DNA Ladder, ready-to-use.......................359 

O’GeneRuler Low Range DNA Ladder ......................................359 

O’GeneRuler Ultra Low Range DNA Ladder, ready-to-use ..........359 

O’RangeRuler 5 bp DNA Ladder, ready-to-use..........................366 

O’RangeRuler 10 bp DNA Ladder, ready-to-use........................366 



O’RangeRuler 100+500 bp DNA Ladder, ready-to-use .............366 

O’RangeRuler 100 bp DNA Ladder, ready-to-use......................366 



ZipRuler Express DNA Ladder Set, ready-to-use .......................365 

RNA ............................................................................................386 

RiboRuler High Range RNA Ladder ..........................................386 

RiboRuler High Range RNA Ladder, ready-to-use ......................386 

RiboRuler Low Range RNA Ladder ...........................................386 

RiboRuler Low Range RNA Ladder, ready-to-use ......................386 

Lambda DNA ...................................................................................291 


Lambda DNA (dam – , dcm – ) ...............................................................291 

Lambda DNA/EcoRI+HindIII Marker, 3 ...............................................368 

Lambda DNA/EcoRI+HindIII Marker, 3, ready-to-use ...........................368 

Lambda DNA/EcoRI Marker, 1 ...........................................................368 

Lambda DNA/HindIII Marker, 2 ..........................................................368 

Lambda DNA/HindIII Marker, 2, ready-to-use .....................................368 

Lambda Exonuclease .......................................................................260 

LguI .......................................................................................... 61, 124 

Ligases, Ligation 


T4 DNA Ligase .............................................................................239 

T4 RNA Ligase .............................................................................240 

Loading Dyes ............................................................................372-373 

2X RNA Loading Dye ....................................................................373 

6X DNA Loading Dye ....................................................................373 

6X DNA Loading Dye & SDS Solution .............................................373 

6X MassRuler DNA Loading Dye ...................................................373 

6X Orange DNA Loading Dye ........................................................373 

6X TriTrack DNA Loading Dye ........................................................373 

LO (Labeled Oligonucleotide Test) .........................................4, 8, 77, 236 

Lsp1109I................................................................................... 21, 124 

LweI ................................................................................................125 

M 

M13mp18 

Restriction Map and Sites ......................................................437-438 

M13mp19 


M13/pUC Primers 

M13/pUC reverse sequencing primer (-26), 17-mer .......................416 


M13/pUC sequencing primer (-20), 17-mer ...................................416 



MaeII ...............................................................................................149 

Maps 

M13mp18, M13mp19 ..................................................................437 

pACYC177...................................................................................453 

pACYC184...................................................................................455 

pBluescript II KS(+/-), pBluescript II SK(+/-) ...................................449 

pBR322 .......................................................................................439 

pJET1.2 ......................................................................................432 

pTZ19R, pTZ19U .........................................................................445 

pTZ57R .......................................................................................447 

pUC18, pUC19 .............................................................................441 

pUC57.........................................................................................443 

X174 ........................................................................................435 

Markers 

DNA .....................................................................................368-371 

Lambda DNA/EcoRI+HindIII Marker, 3......................................368 

Lambda DNA/EcoRI+HindIII Marker, 3, ready-to-use .................368 

Lambda DNA/EcoRI Marker, 1 .................................................368 

Lambda DNA/HindIII Marker, 2 ................................................368 

Lambda DNA/HindIII Marker, 2, ready-to-use ...........................368 

pBR322 DNA/BsuRI (HaeIII) Marker, 5 ......................................368 

pUC19 DNA/MspI (HpaII) Marker, 23 ........................................368 

pUC19 DNA/MspI (HpaII) Marker, 23, ready-to-use ...................368 

X174 DNA /BsuRI (HaeIII) Marker, 9 ......................................368 

X174 DNA /BsuRI (HaeIII) Marker, 9, ready-to-use .................368 

MassRuler DNA Ladder Mix, ready-to-use .........................................362 

MassRuler DNA Loading Dye, 6X.......................................................373

MassRuler Express Forward DNA Ladder Mix, ready-to-use ................362 

MassRuler Express HR Forward DNA Ladder, ready-to-use .................362 

MassRuler Express HR Reverse DNA Ladder, ready-to-use .................362 

MassRuler Express LR Forward DNA Ladder, ready-to-use ..................362 

MassRuler Express LR Reverse DNA Ladder, ready-to-use ..................362 

MassRuler Express Reverse DNA Ladder Mix, ready-to-use ................362 

MassRuler High Range DNA Ladder, ready-to-use ..............................362 

MassRuler Low Range DNA Ladder, ready-to-use...............................362 

MauBI ....................................................................................... 46, 125 

Maxima H Minus Double-Stranded cDNA Synthesis Kit .......................286 

MbiI .......................................................................................... 32, 125 

MboI ......................................................................................... 46, 126 

MboII......................................................................................... 47, 126 

Media ....................................................................................... See FastMedia 

Media, Commonly Used ....................................................................467 

Melting Temperature of Duplex DNA and Oligonucleotides ..................465 

Mesophilic DNA Polymerases ............................................................244 

Methylation 

CpG176 

Dam ............................................................................................174 

Dcm ............................................................................................175 

Digestion of Methylated DNA ........................................................173 

EcoBI ..........................................................................................179 

EcoKI...........................................................................................179 

MfeI .......................................................................................... 47, 129 

MgCl 2 , 25 mM .................................................................................421 

Micrococcal Nuclease ......................................................................268 

MjaIV ..............................................................................................122 

MlsI........................................................................................... 49, 127 

MluI .......................................................................................... 47, 127 

MlyI ........................................................................................... 48, 143 

MnlI .......................................................................................... 48, 127 

Modified Nucleotides 

Aminoallyl-dUTP ..........................................................................414 

Aminoallyl-UTP ............................................................................414 

Biotin-11-dUTP ............................................................................414 

dm 4 CTP .......................................................................................415 

dm 5 CTP .......................................................................................415 

dm 6 ATP .......................................................................................415 

Fluorescein-12-dUTP ...................................................................413 

Modifying Enzymes 

Activity of DNA/RNA modifying enzymes in Fermentas buffers .............274 

Quality Control Assays ..................................................................236 

Storage and Shipping ...................................................................237 

Mph1103I ................................................................................. 54, 128 

MreI .......................................................................................... 48, 128 

MscI .......................................................................................... 49, 127 

MseI.......................................................................................... 49, 151 

MslI........................................................................................... 49, 140 

MspI ......................................................................................... 50, 128 

MssI .......................................................................................... 50, 129 

Multiple Cloning Sites 

Double Digestion of pUC19 MCS ...................................................170 

M13mp18, M13mp19 ..................................................................437 

pBluescript II KS(-), pBluescript II KS(+) .........................................450 

pJET1.2 ......................................................................................432 

pTZ19R, pTZ19U .........................................................................445 

pTZ57R .......................................................................................447 

pUC18, pUC19 .............................................................................441 

pUC57.........................................................................................443 

Multiple Displacement Amplification (MDA). .........See Strand Displacement 

MunI ......................................................................................... 47, 129 

Mva1269I .................................................................................. 51, 130 

MvaI .......................................................................................... 50, 129 

MwoI ...............................................................................................123 

N 

NaeI .......................................................................................... 51, 135 

Nb.Bpu10I .......................................................................................155 

Nb.Mva1269I...................................................................................156 

NBT ................................................................................................426 

NciI ............................................................................................. 51, 89 

NcoI .......................................................................................... 52, 130 

NdeI .......................................................................................... 52, 131 

Neoschizomers ................................................................................171 

Newly Generated Recognition Sites Resulting from 

Fill-in of 5’-overhang and Self-ligation ...........................................200 

Ligation of Blunt DNA Ends ...........................................................180 

Ligation of Protruding Compatible DNA Ends ..................................190 

Removal of 3’-overhang and Self-ligation ......................................204 

NheI .......................................................................................... 52, 131 

Nicking Enzymes ..............................................................................155 

NlaIII ......................................................................................... 53, 120 

NlaIV ......................................................................................... 53, 100 

NmuCI ....................................................................................... 53, 132 

NoLimits Custom DNA Ladders .........................................................370 

NoLimits Custom DNA Ladders Service .............................................370 

NoLimits Individual DNA Fragments ...................................................370 

Nondenaturing RNA Electrophoresis ..................................................390 

Non-specific Nuclease and Cross-contamination Assay ........................77 

NotI ........................................................................................... 54, 132 

NruI............................................................................................. 54, 98 

NsbI .......................................................................................... 41, 132 

NsiI ........................................................................................... 54, 128 

NspI .......................................................................................... 55, 153 

Nt.Bpu10I ........................................................................................156 

NTP Set ...........................................................................................411 

Nucleases 

DNase I, RNase-free .....................................................................255 

Endonuclease IV, E.coli (Endo IV) ...................................................256 

Endonuclease V, T.maritima (Endo V) ..............................................257 

Exonuclease I (Exo I) .....................................................................258 

Exonuclease III (Exo III) ..................................................................259 

Lambda Exonuclease ...................................................................260 

Micrococcal Nuclease ..................................................................268 

RNase A, DNase and Protease-free ....................................... 262, 313 

RNase A/T1 Mix ...........................................................................264 

RNase H ......................................................................................266 

RNase I .......................................................................................265 

RNase T1 ....................................................................................263 

S1 Nuclease ................................................................................269 

Nucleic Acids, Size and MW ..............................................................465 

Nucleotides .....................................................................................405 

dNTP Set .....................................................................................408 

Mixes ..........................................................................................408 

dNTP/dUTP Mix ......................................................................408 

dNTP Mix, 2 mM each ............................................................408 

dNTP Mix, 10 mM each ..........................................................408 

dNTP Mix, 25 mM each ..........................................................408 


Aminoallyl-dUTP .....................................................................414 

Aminoallyl-UTP ......................................................................414 


490 

Biotin-11-dUTP ......................................................................414 

dm 4 CTP .................................................................................415 

dm 5 CTP .................................................................................415 

dm 6 ATP .................................................................................415 

Fluorescein-12-dUTP .............................................................413 

Number of Recognition Sites in DNA Molecules ..................................457 

O 

OEM................................................................................................... IX 

O’GeneRuler 1 kb DNA Ladder, ready-to-use .....................................359 

O’GeneRuler 1 kb Plus DNA Ladder, ready-to-use ..............................359 

O’GeneRuler 50 bp DNA Ladder, ready-to-use ...................................359 

O’GeneRuler 100 bp DNA Ladder, ready-to-use .................................359 

O’GeneRuler 100 bp Plus DNA Ladder, ready-to-use ..........................359 

O’GeneRuler DNA Ladder Mix, ready-to-use ......................................359 

O’GeneRuler Express DNA Ladder, ready-to-use ................................359 

O’GeneRuler Low Range DNA Ladder, ready-to-use ...........................359 

O’GeneRuler Ultra Low Range DNA Ladder, ready-to-use ....................359 

Oligo(dT) 18 Primer ............................................................................417 

Oligonucleotides ..............................................................................416 

OliI ............................................................................................ 16, 133 

Orange DNA Loading Dye, 6X ...........................................................373 

O’RangeRuler 5 bp DNA Ladder, ready-to-use ...................................366 

O’RangeRuler 10 bp DNA Ladder, ready-to-use .................................366 



O’RangeRuler 100+500 bp DNA Ladder, ready-to-use .......................366 

O’RangeRuler 100 bp DNA Ladder, ready-to-use ...............................366 



Ordering Iinformation .........................................................................VIII 

ORF ................................................................................................512 

P 

PacI .......................................................................................... 55, 133 

pACYC177 Restriction Map and Sites ................................................453 

pACYC184 Restriction Map and Sites ................................................455 

PaeI .......................................................................................... 66, 133 

PagI .......................................................................................... 31, 134 

PasI ................................................................................................134 

Patents............................................................................................502 

PauI ................................................................................................134 

pBluescript DNA Restriction Map and Sites ........................................449 

pBR322 DNA ...................................................................................291 

Restriction Map and Sites .............................................................439 

pBR322 DNA/BsuRI (HaeIII) Marker, 5 ................................................368 

PciI .................................................................................................137 

PCR, Analysis 

Agaroses .....................................................................................427 

DNA Ladders and Markers ............................................................354 

PCR, Clean-up 

Agarase .......................................................................................319 

Alkaline Phosphatases ...........................................................242-243 

Exonuclease I ...............................................................................258 



PCR, Cloning 





T4 DNA Polymerase .....................................................................249 

T4 Polynucleotide Kinase ..............................................................243 

PCR, DNA Purification 



GeneJET PCR Purification Kit ........................................................316 



Glycogen .....................................................................................423 

Proteinase K ................................................................................270 

RNases .................................................................262-265, 313-316 

PCR, Ladders and Markers 

FastRuler DNA Ladders ................................................................364 

GeneRuler DNA Ladders ...............................................................358 

O’GeneRuler DNA Ladders ............................................................358 

O’RangeRuler DNA Ladders ..........................................................366 

ZipRuler DNA Ladder Set ..............................................................365 

PCR, Nucleotides 

dNTP Set and Mixes .....................................................................408 


PCR, qPCR and RT-PCR 

Digestion of PCR Products ...................................................72, 73-76 

PCR, Sequencing 

Alkaline Phosphatases ...........................................................242-243 

Exonuclease I (Exo I) .....................................................................258 

PCR, Single-stranded PCR Product 

Lambda Exonuclease ...................................................................260 

PdiI ........................................................................................... 51, 135 

PdmI ......................................................................................... 55, 135 

PfeI ........................................................................................... 69, 135 

Pfl23II ....................................................................................... 28, 136 

PflMI ......................................................................................... 56, 152 

PfoI ........................................................................................... 56, 136 

Phage and Plasmid DNA 

FX174 RF1 DNA ..........................................................................291 

Lambda DNA ...............................................................................291 

Lambda DNA (dam – , dcm – ) ...........................................................291 

M13mp18 ...................................................................................437 

M13mp19 ...................................................................................437 

pACYC177...................................................................................453 

pACYC184...................................................................................455 

pBluescript II KS(+/-) ....................................................................449 

pBluescript II SK(+/-) ....................................................................449 

pBR322 .......................................................................................439 

pJET1.2 DNA ...............................................................................432 

pTZ19R .......................................................................................445 

pTZ19U .......................................................................................445 

pTZ57R .......................................................................................447 

pUC18.........................................................................................441 

pUC19.........................................................................................441 

pUC57.........................................................................................443 

X174 ........................................................................................435 

phi29 DNA Polymerase ....................................................................245 

Phosphatases 


Phosphorylation 

T4 Polynucleotide Kinase (T4 PNK) ................................................243 

pJET1.2 ..........................................................................................432 

pJET1.2 Forward Sequencing Primer, ................................................416 

pJET1.2 Reverse Sequencing Primer .................................................416 

Plasmid DNA Purification 

GeneJET Plasmid Maxiprep Kit ......................................................306

GeneJET Plasmid Midiprep Kit ......................................................305 


PmeI ...............................................................................................129 

PmlI .......................................................................................... 56, 112 

Polyacrylamide Gels ................................................................. 375, 391 

Polymerases 

Bsm DNA Polymerase, Large Fragment .........................................253 

DNA Polymerase I ........................................................................246 

Klenow Fragment .........................................................................247 

Klenow Fragment, exo – .................................................................248 

phi29 DNA Polymerase.................................................................245 



Terminal Deoxynucleotidyl Transferase (TdT) ..................................251 

Ppu21I ...................................................................................... 25, 137 

PpuMI ....................................................................................... 57, 138 

Primers ...........................................................................................416 

Proteinase K (recombinant), PCR grade .............................................270 

Protocols and Recommendations for 

Conventional Restriction Enzymes .................................................158 



FastDigest Restriction Enzymes .......................................................72 


Molecular Cloning ........................................................................294 

Molecular Labeling and detection ..................................................349 



Protocols (Protocol Index) .................................................................500 

PscI ................................................................................................137 

PshAI .......................................................................................... 57, 92 

PsiI ............................................................................................. 57, 82 

Psp5II........................................................................................ 57, 138 

Psp1406I .................................................................................. 14, 138 

PspFI.................................................................................................58 

PspOMI .............................................................................................99 

PstI ........................................................................................... 58, 138 

PsuI .......................................................................................... 58, 139 

PsyI ........................................................................................... 59, 139 

PteI ...................................................................................................33 

pTZ19R DNA ...................................................................................292 

pTZ19R, pTZ19U, pTZ57R 


pUC18 DNA .....................................................................................292 

pUC18, pUC19, pUC57 


pUC19 DNA .....................................................................................292 

pUC19 DNA/MspI (HpaII) Marker, 23..................................................368 

pUC19 DNA/MspI (HpaII) Marker, 23, ready-to-use .............................368 

pUC57 DNA .....................................................................................292 

Purification 

Genomic DNA 

GeneJET Genomic DNA Purification Kit ....................................310 

Genomic DNA Purification Kit ..................................................311 

PCR Products 

GeneJET PCR Purification Kit ..................................................316 

Plasmid DNA 

GeneJET Plasmid Maxiprep Kit ................................................306 

GeneJET Plasmid Midiprep Kit ................................................305 

GeneJET Plasmid Miniprep Kit ................................................304 

RNA 

GeneJET RNA Purification Kit ..................................................307 

PvuI .......................................................................................... 59, 139 

PvuII .......................................................................................... 59, 140 

Pyrophosphatase, Inorganic (from yeast) ............................................272 

Q 

Quality Control Assays .............................................................8, 77, 236 

R 

Radioisotopes ..................................................................................462 

Random Displacement Amplification (RDA)...........See Strand Displacement 

Random Hexamer Primer ..................................................................417 

Random Primer 

Exo-Resistant Random Primer .......................................................417 

Random Hexamer Primer ..............................................................417 

Rapid DNA Ligation Kit .....................................................................288 

Reagents .........................................................................................419 

Recognition Sites in DNA Molecules ..................................................457 

Recomendations for 

Conventional Restriction Enzymes .................................................158 

FastDigest Restriction Enzymes .......................................................72 

Recommendations for 





Molecular Labeling and Detection .................................................349 



Recommendations for Loading 


RNA Ladders ...............................................................................390 

Reducing Agent ...............................................................................420 

Relaxation of Specificity (Star Activity) ................................................172 


Activity Assay ............................................................................. 8, 77 

Alphabetic Lists of 

Commercially Available Restriction Enzymes ............................205 

Conventional Restriction Enzymes .............................................78 

FastDigest Restriction Enzymes ...................................................9 

Recognition Sequences ..........................................................217 

Classification of Restriction Enzymes .............................................171 

Digestion of Methylated DNA ........................................................173 

Navigation Guide ..............................................................................5 

Newly Generated Cleavage Sites ...................................................180 

Product Description .................................................................. 13, 82 

Protocols and Recommendations ............................................ 72, 158 

Reaction Conditions ............................................................... 73, 160 

Selection Guides ..........................................................................205 

Star Activity .................................................................................172 

Storage and Shipping ................................................................. 8, 77 

Troubleshooting Guide ..................................................................229 

RiboLock RNase Inhibitor ..................................................................271 

RiboRuler RNA Ladders 

High Range ..................................................................................386 

High Range, ready-to-use .............................................................386 

Low Range ..................................................................................386 

Low Range, ready-to-use..............................................................386 

RNA Electrophoresis 

Agarose .......................................................................................427 

Ladders .......................................................................................386 

Protocols and Recommendations ..................................................390 


492 

RNAi ...............................................................................................512 

RNA Ladders ...................................................................................386 

RNA Loading Dye .............................................................................388 

RNA Polymerases 

SP6 RNA Polymerase ...................................................................331 

T3 RNA Polymerase .....................................................................331 


RNA Purification 



Glycogen .....................................................................................423 

RNase A, DNase and Protease-free ........................................... 262, 313 

RNase A/T1 Mix ...............................................................................264 

RNase H ..........................................................................................266 

RNase I ...........................................................................................265 

RNase T1 ........................................................................................263 

RNA Sizing ......................................................................................386 

RruI ...................................................................................................54 

RsaI .......................................................................................... 60, 140 

RseI .......................................................................................... 49, 140 

RsrII .......................................................................................... 60, 106 

RT-PCR, Analysis on Gels 

Agarose .......................................................................................427 


Electrophoresis Buffers .................................................................371 

RiboRuler RNA Ladders ................................................................386 

RT-PCR, Clean-up 

Agarase .......................................................................................319 



RT-PCR, Cloning 





T4 Polynucleotide Kinase (T4 PNK) ................................................243 

RT-PCR, Enzymes 


Thermophilic DNA Polymerases .................................................. .See PCR 

RT-PCR, Nucleotides 

dNTP Mixes .................................................................................408 

dNTP Set .....................................................................................408 


RT PCR, Primers for cDNA Synthesis 


Oligo(dT) 18 Primer.........................................................................417 


RT-PCR, Purification of RT-PCR Products .................. See RT-PCR, Clean-up 

RT-PCR, RNA Purification 



Glycogen, RNA grade ...................................................................423 

RT-PCR, RNase Inhibitor 


RT-PCR, Second Strand cDNA Synthesis 


RNase H ......................................................................................266 

S 

S1 Nuclease ....................................................................................269 

SacI .......................................................................................... 60, 141 


SacII................................................................................................106 

SalI ........................................................................................... 61, 141 

SanDI ................................................................................................61 

SapI .......................................................................................... 61, 124 

SaqAI ................................................................................................49 

SatI ........................................................................................... 40, 142 

Sau3AI ........................................................................................ 62, 99 

Sau96I ...................................................................................... 62, 105 

SbfI ........................................................................................... 62, 143 

ScaI .......................................................................................... 63, 142 

SchI .......................................................................................... 48, 143 

ScrFI ........................................................................................... 63, 91 

SdaI .......................................................................................... 62, 143 

SduI .......................................................................................... 30, 143 

Second Strand cDNA Synthesis 


RNase H ......................................................................................266 

Selection Guides for 

DNA/RNA Polymerases 

Mesophilic DNA Polymerases ..................................................244 


Ligases .......................................................................................238 


Molecular Labeling and Detection .................................................342 

Nucleases 

Deoxyribonucleases (DNases) .................................................254 

Ribonucleases (RNases)..........................................................261 

Sugar Nonspecific Nucleases ..................................................268 


Phosphatases and Kinase .............................................................241 

RNA Ladders ...............................................................................386 

Sensitivity to Methylation 

CpG Methylation ..........................................................................176 

Dam Methylation ..........................................................................174 

Dcm Methylation ..........................................................................175 

EcoKI and EcoBI Methylation .........................................................179 

Sequencing Primers 






pJET1.2 forward sequencing primer, 23-mer .................................416 

pJET1.2 reverse sequencing primer, 24-mer .................................416 

SP6 promoter sequencing primer, 18-mer .....................................416 

SP6 promoter sequencing primer, 24-mer .....................................416 

T3 promoter sequencing primer, 17-mer .......................................416 



SexAI ................................................................................................63 

SfaAI ......................................................................................... 19, 144 

SfaNI ......................................................................................... 64, 125 

SfcI ............................................................................................. 64, 90 

SfiI ............................................................................................ 64, 144 

SfoI .................................................................................................116 

SgeI ................................................................................................145 

SgrDI...............................................................................................145 

SgsI .......................................................................................... 18, 145 

Silica Bead DNA Gel Extraction Kit .....................................................318 

siRNA ..............................................................................................512 

siRNA Analysis on Gel ......................................................................387 

Site Preferences by Restriction Endonucleases ..................................171

SI Unit Prefixes ................................................................................464 

SmaI ......................................................................................... 65, 146 

SmiI .......................................................................................... 67, 146 

SmlI ................................................................................................147 

SmoI ...............................................................................................147 

SnaBI ........................................................................................ 65, 114 

Sodium Acetate Solution, 3 M, pH 5.2 ...............................................421 

SP6 Promoter Sequencing Primers ...................................................416 

SP6 RNA Polymerase .......................................................................331 

SpeI ............................................................................................ 65, 89 

SphI .......................................................................................... 66, 133 

Sse232I ..........................................................................................128 

SsiI ........................................................................................... 14, 147 

SspDI ..............................................................................................148 

SspI .......................................................................................... 66, 148 

Star Activity .....................................................................................172 

Step Ladders ...................................................................................366 

Stock Solutions, Commonly Used ......................................................467 

Strand Displacement 

Bsm DNA Polymerase, Large Fragment .........................................253 


phi29 DNA Polymerase.................................................................245 


StuI ........................................................................................... 66, 114 

StyI ........................................................................................... 67, 114 

SwaI.......................................................................................... 67, 146 

T 

T3 promoter sequencing primer, 17-mer ...........................................416 


T3 RNA Polymerase .........................................................................331 

T4 DNA Ligase .................................................................................239 

T4 DNA Polymerase .........................................................................249 

T4 Polynucleotide Kinase (T4 PNK) ....................................................243 

T4 RNA Ligase .................................................................................240 

T7 DNA Polymerase .........................................................................250 


T7 RNA Polymerase .........................................................................330 

TaaI ........................................................................................... 67, 149 

TAE Buffer, 50X ................................................................................371 

TaiI ....................................................................................................68 

Tail ..................................................................................................149 

TaqI ........................................................................................... 68, 149 

TasI ........................................................................................... 70, 150 

TatI ........................................................................................... 68, 150 

TauI ........................................................................................... 69, 150 

TBE Buffer, 10X ...............................................................................371 

TdT (Terminal Deoxynucleotidyl Transferase) ......................................251 

Temperature Dependence of pH 

for Commonly Used Buffers ..........................................................466 

of 50 mM Tris-HCl Solutions .........................................................465 

Terminal Deoxynucleotidyl Transferase (TdT) ......................................251 

TfiI ............................................................................................ 69, 135 

TopVision Agarose ............................................................................427 

TopVision Low Melting Point Agarose.................................................427 

Trademarks .....................................................................................503 

TranscriptAid T7 High Yield Transcription Kit .......................................328 

Transcription in vitro 


RNA Ladders ...............................................................................386 

SP6 RNA Polymerase ...................................................................331 



TranscriptAid T7 High Yield Transcription Kit ...................................328 

Transcription Promoter Primers .........................................................416 

Transferase .....................................................................................251 

TransformAid Bacterial Transformation Kit ..........................................289 

Troubleshooting Guide for 

DNA Digestion .............................................................................229 





Tru1I ......................................................................................... 69, 151 

TscAI......................................................................................... 70, 151 

Tsp45I ............................................................................................132 

Tsp509I .................................................................................... 70, 150 

TspRI ........................................................................................ 70, 151 

Tth111I ...........................................................................................139 

U 

Uracil-DNA Glycosylase (UDG, UNG) ..................................................273 

UTP .................................................................................................412 

V 

Van91I ...................................................................................... 56, 152 

Viral DNA/RNA Purification Kit (IVD) ...................................................315 

VspI .......................................................................................... 19, 152 

W 

Water, DEPC-treated ........................................................................420 

Water, nuclease-free ........................................................................420 

WELQut Protease .............................................................................285 

Whole Genome Amplification (WGA) .......................................... 245, 253 

X 

XagI .......................................................................................... 38, 152 

XapI .......................................................................................... 70, 153 

XbaI .......................................................................................... 71, 153 

XceI .......................................................................................... 55, 153 

X-Gal ..............................................................................................424 

X-Gal Solution ..................................................................................424 

X-Gluc .............................................................................................425 

XhoI .......................................................................................... 71, 154 

XmaI ...............................................................................................105 

XmaJI........................................................................................ 20, 154 

XmiI .......................................................................................... 13, 154 

XmnI ...............................................................................................135 

Z 

ZipRuler Express DNA Ladder Set, ready-to-use .................................365 


494 

Protocol Index 

Thermo Scientific FastDigest & Conventional Restriction Enzymes 

1.1. Fast DNA digestion ..................................................................72 

1.2. Reaction set-up for digestion of multiple DNA samples ..............72 

1.3. Double and multiple digestion of DNA .......................................72 

1.4. Scaling up DNA digestion reaction ............................................72 

1.5. DNA digestion .......................................................................158 

1.6. Digestion of PCR products .....................................................158 

1.7. Setting up double digestion ....................................................158 

1.7.1. Double digestion with FastDigest enzymes .....................158 

1.7.2. DoubleDigest Engine ....................................................158 

1.7.3. Double digestion in the universal Tango Buffer ...............158 

1.7.4. Double digestion in the Five Buffer System .....................158 

1.7.5. Digestion with enzymes having different 

optimum temperatures ...........................................................158 

1.7.6. Sequential digestion .....................................................158 

1.8. Stability during prolonged incubation ......................................158 

1.9. Dilution of restriction enzymes................................................158 

1.10. Partial digestion of DNA .......................................................158 

1.11. Digestion of agarose-embedded DNA ...................................159 

1.12. Inactivation of restriction enzymes ........................................159 

Thermo Scientific DNA/RNA Modifying Enzymes 

2.1. Activity of DNA/RNA modifying enzymes 

in Thermo Scientific buffers ..........................................................274 

2.2. Dilution of DNA/RNA modifying enzymes .................................275 

Thermo Scientific Molecular Cloning Products 

3.1. DNA ligation ..........................................................................294 

3.1.1. Sticky-end ligation ........................................................294 

3.1.2. Blunt-end ligation .........................................................294 

3.1.3. Self-circularization of linear DNA ...................................294 

3.1.4. Linker ligation ..............................................................294 

3.1.5. Analysis of ligation products by agarose 

gel electrophoresis .................................................................294 

3.1.6. Control reaction for T4 DNA Ligase activity .....................294 

3.2. DNA blunting ........................................................................295 

3.2.1. Blunting of 5’- or 3’-overhangs with 

T4 DNA Polymerase ...............................................................295 

3.2.2. Fill-in of 5’-overhangs with Klenow Fragment, exo – ...........295 

3.2.3. Removal of 3’- and 5’-overhangs 

with S1 Nuclease ...................................................................295 

3.3. Phosphorylation of DNA .........................................................295 

3.4. Dephosphorylation of DNA and RNA .......................................295 

3.4.1. Dephosphorylation of DNA and RNA ..............................295 

3.4.2. Fast simultaneous plasmid vector linearization 

and dephosphorylation ...........................................................295 

3.5. Removal of tags from recombinant proteins 

using WELQut Protease ................................................................295 

3.5.1. Optimization of WELQut Protease cleavage .....................295 

3.5.2. Cleavage of fusion proteins in solution ...........................296 

3.5.3. Cleavage of fusion proteins during 

affinity purification ..................................................................296 

3.6. Transformation ......................................................................296 

3.7. Cultivation of E.coli ................................................................296 

3.7.1. Preparation of E.coli culture glycerol stocks ...................296 

3.7.2. Preparation of X-Gal/IPTG LB agar plates 

for blue/white colony screening ...............................................296 

3.8. Analysis of recombinant clones ..............................................296 


Thermo Scientific Nucleic Acid Purification Products 

4.1. Purification of genomic DNA from mouse tails 

with Proteinase K .........................................................................322 

4.2. Purification of DNA from cultured eukaryotic cells 

with Proteinase K .........................................................................322 

4.3. Casting high quality agarose gels ...........................................322 

4.4. Recovery of DNA from LM agarose gels with Agarase ..............322 

4.5. Nucleic acid precipitation from diluted solutions 

with Glycogen ..............................................................................323 

4.6. Removal of template DNA after in vitro transcription ................323 

4.7. Removal of genomic DNA from RNA preparations ....................323 

4.8. PCR product clean-up prior to sequencing ..............................323 

4.9. Phenol/chloroform extraction and ethanol precipitation ............323 

Thermo Scientific in vitro Transcription Products 

5.1. DNA template preparation for in vitro transcription...................335 

5.2. Avoiding RNase contamination ...............................................335 

5.3. in vitro transcription ...............................................................335 

5.4. Synthesis of radiolabeled RNA probes 

of high specific activity .................................................................335 

5.5. Purification of RNA transcripts ................................................336 

5.6. Evaluation of transcription reaction products ...........................336 

Thermo Scientific Molecular Labeling & Detection Products 

6.1. DNA/RNA end labeling ...........................................................349 

6.1.1. DNA 5’-end labeling by T4 PNK 

in the exchange reaction.........................................................349 

6.1.2. DNA/RNA 5’-end labeling by 

T4 PNK in the forward reaction ...............................................349 

6.1.3. Radiolabeling of RNA Ladders by T4 PNK .......................349 

6.1.4. DNA 3’-end labeling by fill-in of 5’-overhangs ................349 

6.1.5. DNA and oligonucleotide 3’-end labeling by tailing .........349 

6.1.6. RNA 3’-end labeling by ligation .....................................350 

6.2. Random-primed labeling........................................................350 

6.2.1. Radioactive random-primed DNA labeling ......................350 

6.2.2. Non-radioactive random-primed DNA labeling ................350 

6.3. DNA labeling by nick-translation .............................................350 

6.3.1. Radioactive DNA labeling by nick-translation ..................350 

6.3.2. Non-radioactive DNA labeling by nick-translation ............350 

6.4. RNA labeling by in vitro transcription ......................................350 

6.4.1. Synthesis of radiolabeled RNA probes 

of high specific activity ...........................................................350 

6.5. Synthesis of labeled cDNA .....................................................350 

6.5.1. Synthesis of cDNA probes 

with high specific radioactivity .................................................350 

6.5.2. Synthesis of non-radioactively labeled cDNA ..................351 

6.6. Southern Blotting ..................................................................351 

Thermo Scientific DNA Electrophoresis Products 

7.1. General recommendations for DNA electrophoresis .................374 

7.1.1. Recommendations for accurate 

in-gel DNA quantification ........................................................374 

7.2. Preparation of agarose gels for DNA electrophoresis ...............374 

7.2.1. Non-denaturing agarose gel electrophoresis ..................374 

7.2.2. Alkaline agarose gel electrophoresis ..............................375 

7.3. Preparation of gels for PAGE ..................................................375 

7.3.1. Non-denaturing PAGE ...................................................375 

7.3.2. Denaturing polyacrylamide/urea gel electrophoresis .......375

7.4. Preparation of DNA samples for electrophoresis ......................376 

7.4.1. Preparation of DNA samples for conventional 

DNA electrophoresis ...............................................................376 

7.4.2. Preparation of DNA samples for denaturing 

polyacrylamide/urea gel electrophoresis ..................................376 

7.4.3. Preparation of DNA samples for denaturing 

alkaline agarose gel electrophoresis ........................................376 

7.4.4. Preparation of DNA samples containing 

DNA binding proteins ..............................................................376 

7.5. Preparation of DNA ladders/markers for electrophoresis ..........376 

7.6. Labeling of DNA ladders/markers ...........................................376 

7.7. Separation of Express DNA ladders in different 

electrophoresis conditions ............................................................377 

Thermo Scientific RNA Electrophoresis Products 

8.1. General recommendations for RNA electrophoresis .................390 

8.2. Preparation of RNA ladders for electrophoresis ........................390 

8.3. Preparation of RNA samples for electrophoresis ......................390 

8.4. Preparation of gels for RNA electrophoresis ............................390 

8.4.1. Non-denaturing agarose gels ........................................390 

8.4.3. Denaturing glyoxal/DMSO gels in sodium 

phosphate buffer ....................................................................390 

8.4.4. Denaturing polyacrylamide/urea gels 

in TBE buffer ..........................................................................391 


496 

Patents 

The purchase of below listed products includes a non-transferable license 

to use these products for the purchaser’s internal research. All other 

commercial uses of these products, including without limitation product 

use for diagnostic purposes, resale of product in the original or any 

modified form or product use in providing commercial services require a 

separate license from Fermentas, a subsidiary of Thermo Fisher Scientific. 

For further information on obtaining licenses please contact at 

info.fermentas.lt@thermofisher.com 

Nickases (N.Bpu10I) 

This product or the use of this product is covered by US patent 

No. 6,867,028 and corresponding counterparts. 

Restriction enzymes with altered specificity (Eco57MI) 

This product or the use of this product is covered by US patent 


Nucleic acid purification process (NoLimits fragments, 

DNA ladders) 

Manufacture of this product uses process covered by US patent 


pJET1.2 vector 

This product or the use of this product is covered by US patent application 

US20090042249A1 and corresponding counterparts. 

Protein evolution in vitro method; Mutant Reverse 

Transcriptase technology (RevertAid Premium and Maxima RT) 



Methylation-specific REases (SgeI) 



dsRNA synthesis using Phi6 RNA Replicase 

This product or the use of this product is covered by EP patent 

EP1242586B1 and other patents exclusively licensed to 

Thermo Fisher Scientific Inc. and its subsidiaries. 


Licenses 

Fermentas and/or its affiliates products may be covered by one or more 

licenses. By use of these products you agree to comply with the terms 

and conditions of applicable license: 

Nicking enzyme technology (Nb.Mva1269I) 

This product is licensed under US. Patent No. 7,081,358 or corresponding 

foreign patents. 

Recombinant thermostable DNA polymerase 

from archaebacteria 

This product is licensed under EP patent EP0547920B1, owned by 

New England Biolabs, Inc. 

Homing endonuclease I-SceI 

This product is licensed under one or more US Patent Nos. 

5,474,896, 5,866,361, 5,792,632 or corresponding counterparts. 

Nucleic acid marker ladder for estimating mass 

Purchase of this product includes a limited license under US Patent 

Nos. 5,834,201, 6,680,378, and/or 7,132,520 to use this product for 

internal research purposes only. 

Bsm DNA Polymerase 

This product is patent pending with rights co-owned by K.K. 

DNAFORM and RIKEN, The Institute of Physical and Chemical 

Research. 

PCR Product Clean-up Prior to Sequencing 

(Exonuclease I, Alkaline phosphatases) 

The purchase of this product allows the purchaser to use it for 

preparing amplified DNA fragments under a license from GE 

Healthcare of US Patent Nos. 5,741,676 and 5,756,285 and other 

foreign patents.

Trademarks 

aLICator, CloneJET, DecaLabel, DoubleDigest, DreamTaq, EpiJET, FastAP, 

FastDigest, FastDigest and PureExtreme, FastRuler, GeneJET, GeneRuler, 

InsTAclone, MassRuler, Maxima, NoLimits, O’GeneRuler, O’RangeRuler, 

PageRuler, PageSilver, Phusion, PowerCut, Premium, PureExtreme, 

Replicator, REsearch, REviewer, RevertAid, RiboLock, RiboRuler, Tango, 

TopVision, TranscriptAid, TransformAid, TriTrack, WELQut, ZipRuler are 

trademarks of Thermo Fisher Scientific Inc. and its subsidiaries. 

Cy, Ficoll, and Sephadex are trademarks of GE Healthcare. 

DH5 and DH5F’ are trademarks of Life Technologies, Inc. 

Elugent is a trademark of Calbiochem Novabiochem, Inc. 

LabChip is a trademark of Caliper Life Sciences, Inc. 

Nonidet is a trademark of Shell Chemical Co. 

Norit is a registered trademark of Norit N.V. 

pBluescript is a trademark of Agilent Technologies, Inc. 

Saran Wrap is a trademark of the Dow Chemical Company. 

Triton X-100 is a trademark of Union Carbide Chemicals & Plastics 

Technology Corporation. 

Tween is a trademark of ICI Americas, Inc. 

Whatman is a trademark of Whatman Ltd. 


498 

Notes 


Notes 


500 

Notes 


Notes 


502 

Solutions for PCR and qPCR 

The Thermo Scientific PCR and qPCR portfolio has everything you need for a successful run: reagents, instruments and plastic 

consumables. Our innovative portfolio is assembled by combining the state-of-the-art product lines from ABgene, Finnzymes and 

Fermentas. The products within – born of scientific breakthroughs, themselves – are accelerating discovery in life science, and our 

aim is to continue to develop new tools to increase your efficiency and enhance your effectiveness. 

Solutions for RNAi and Gene Expression 

The Thermo Scientific RNAi and gene expression products encompass the most comprehensive portfolio of tools for gene 

knockdown and over-expression. Our innovative product portfolio combines state-of-the-art product lines from Dharmacon and 

Open Biosystems. Find industry-leading siRNA, shRNA, microRNA, cDNA and ORF reagents as well as a complete suite of controls 

and solutions for RNAi delivery and screening. 


Find the complete workflow of Thermo Scientific 

PCR and qPCR products, including reagents, 

instruments and plastic consumables at 

www.thermoscientific.com/pcr 

From single gene analysis to genome-scale screens, 

find your RNAi and Gene Expression solutions at 

www.thermoscientific.com/silence

Contact details 

North America 

Technical Services 

ts.molbio@thermofisher.com 

Customer Services 

cs.molbio@thermofisher.com 

Ordering Information 


Europe and Asia 

Technical Services 

ts.molbio.eu@thermofisher.com 

Customer Services 

cs.molbio.eu@thermofisher.com 

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Thermo Scientific Molecular Biology Solutions 2012 - 2013 - Biocenter

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