EPICENTRE Forum 14-2
EPICENTRE Forum 14-2
EPICENTRE Forum 14-2
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Volume <strong>14</strong>-2<br />
Featuring:<br />
New Systems for RNA<br />
and cDNA Studies<br />
Introducing the ExactStart <br />
Full-Length cDNA Library<br />
Cloning Kit for Obtaining<br />
Full-Length cDNAs and<br />
Accurate Mapping of<br />
Transcript Ends<br />
Achieve Fast Real-Time PCR<br />
Results With the New PCR<br />
MasterMix Kit<br />
Extract PCR-Ready Plant Genomic<br />
DNA Using a Simple 8-Minute,<br />
1-Tube Protocol<br />
Detect More Genes With the New<br />
TargetAmp Kit Designed for the<br />
Illumina ® System<br />
Cover Photo: La Jolla, CA<br />
Photograph by: Dilara Begum,<br />
<strong>EPICENTRE</strong> Biotechnologies, Madison, WI
Be certain that ZERO<br />
DNA remains<br />
NEW! <strong>EPICENTRE</strong> Biotechnologies’<br />
Baseline-ZERO DNase digests doubleand<br />
single-stranded DNA more completely<br />
than any other product on the market.<br />
qPCR Demonstrating DNA Contaminants<br />
• Removes genomic DNA from RNA prior to<br />
RT-PCR.<br />
• Prepares target RNA for cDNA in microarray<br />
or expression analysis.<br />
• Eliminates the DNA template following<br />
in vitro RNA synthesis with T7, T3, or SP6<br />
Phage RNA Polymerases.<br />
• Removes ssDNA and dsDNA from viral<br />
RNA.<br />
• Eliminates genomic DNA from RNA for<br />
microinjection and transfection experiments.<br />
C T<br />
values from real-time PCR of HeLa RNA preparations treated with various<br />
DNases. The lower the C T<br />
value, the greater the amount of residual DNA not<br />
digested by the indicated DNase. Thus, Baseline-ZERO DNase removed all<br />
detectable DNA from the RNA sample. The TaqMan probe assay amplifi ed a<br />
268 bp fragment of beta actin. Samples were run in duplicate.<br />
To order yours, go to www.EpiBio.com<br />
and enter code: BZAX3<br />
• Simple 10 minute heat inactivation.
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
IN THIS ISSUE<br />
ExactStart Full-Length cDNA Library Cloning Kit: A Rapid and Efficient<br />
Method to Synthesize Full-Length cDNA for Cloning and Accurate<br />
Mapping of Transcription Initiation and Polyadenylation Sites. . . . . . . . . 4<br />
The new ExactStart Full-Length cDNA Library Cloning Kit produces full-length cDNA libraries<br />
from a variety of eukaryotic sources. These libraries can be used to study alternate 5′ transcriptional<br />
start and 3′ polyadenylation sites.<br />
Fast Real-Time PCR with a Standard qPCR Thermocycler! . . . . . . . . . . . 6<br />
The TAQXpedite GREEN Real-Time PCR MasterMix Kit enables fast real-time PCR (fast<br />
qPCR) reactions to be performed using standard qPCR instrumentation.<br />
HOW TO CONTACT US<br />
Toll-free in the USA: 800-284-8474<br />
Tel: 608-258-3080 Fax: 608-258-3088<br />
Mail: 726 Post Rd., Madison, WI 53713<br />
Website: www.EpiBio.com<br />
E-mail: forum@EpiBio.com<br />
Outside the USA: contact your local<br />
<strong>EPICENTRE</strong> Distributor.<br />
www.EpiBio.com/distributors.asp<br />
<strong>EPICENTRE</strong> Biotechnologies FORUM<br />
Editor: Simon T. M. Allard, Ph.D.<br />
Graphic Designer: Julie Capadona<br />
Additional Illustrations: Ron Meis<br />
COMPLIMENTARY<br />
Receive each issue of the<br />
<strong>EPICENTRE</strong> <strong>Forum</strong> by subscribing at:<br />
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©2007 <strong>EPICENTRE</strong> ® Biotechnologies All rights<br />
reserved. Publication date: October 2007. Printed in USA.<br />
<strong>EPICENTRE</strong>, Ampligase, DuraScribe, and DuraScript<br />
are registered trademarks and 2-Way, A-Plus, AmpliCap,<br />
AmpliCap-Max, AmpliScribe, AmpliTherm, APex,<br />
A-Plus, ArrayPure, BAC-Tracker, BACMAX, BACMAX96,<br />
BuccalAmp, Catch-All, CircLigase, Colony Fast-Screen,<br />
CopyControl, CopyCutter, EC100, EC100D, EPI300,<br />
EPI400, EasyLyse, End-It, EpiBlue, EpiFOS, Epi-Grids,<br />
EpiLyse, ExactStart, ExtractMaster, EZ::TN, EZ-Tn5,<br />
FailSafe, Fast-Link, Fast-Screen, FosmidMAX, GELase,<br />
HK, Hybridase, HyperMu, IsoTherm, Kool, Long-Read,<br />
MasterAmp, MasterPure, MaxPlax, MessageBOOSTER,<br />
MessageMAX, MessageMuter, MiniV, MonsterScript,<br />
mRNA-ONLY, mScript, Mu-End, NC-45, NTPhos,<br />
Nano-g, OmniCleave, pCC1, pCC1BAC, pCC1FOS,<br />
pCC2FOS, PCRLyse, PeriPreps, PlasmidMAX,<br />
Plasmid-Safe, pEpiFOS, pMOD, pPDM, pWEB,<br />
pWEB-TNC, QuickExtract, R&DNA, Ready-Lyse,<br />
ReadyPreps, RepliPHI, Restricti-Lyse, RetroAmp, RiboScribe,<br />
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SequiTherm EXCEL, SoilMaster, T3-Flash, T7-Flash,<br />
Tagetin, TAQurate, TAQXpedite, TargetAmp, Terminator,<br />
TransforMax, Transposome, Transposomics, TypeOne, and<br />
WaterMaster are trademarks of <strong>EPICENTRE</strong>. RNeasy<br />
is a registered trademark of Qiagen Corp. GeneChip is a<br />
registered trademark of Affymetrix. CodeLink and Cy are<br />
trademarks of GE Healthcare. SuperScript is a registered<br />
trademark of Invitrogen. SYBR is a registered trademark of<br />
Molecular Probes, Invitrogen Detection Technologies. Product<br />
prices and specifications are subject to change without notice.<br />
Easy and Rapid Extraction of Plant DNA Using the QuickExtract Plant<br />
DNA Extraction Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7<br />
The QuickExtract Plant DNA Extraction Solution represents a rapid and efficient way to extract<br />
PCR-ready plant genomic DNA from most plant samples.<br />
Product Data Sheets for <strong>EPICENTRE</strong> Biotechnologies’ Products ........ 9<br />
TargetAmp Nano-g Biotin-aRNA Labeling Kit for the Illumina® System<br />
TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0<br />
EZ-Tn5 Insertion Kit<br />
EZ-Tn5 In-Frame Linker Insertion Kit<br />
EZ-Tn5 Promoter Insertion Kit<br />
EZ-Tn5 Transposon Construction Vectors<br />
QuickExtract Plant DNA Extraction Solution<br />
QuickExtract DNA Extraction Solution 1.0<br />
TAQXpedite PCR System (FAST End-Point)<br />
TAQXpedite GREEN Real-Time PCR MasterMix Kit<br />
Ask Frank, by Fred Hyde and Hank Daum . . . . . . . . . . . . . . . . . . . . . . . 17<br />
Questions about Capping of RNA and in vitro transcription, answered by <strong>EPICENTRE</strong>’s Technical<br />
Consultants.<br />
A Simple Enzymatic Process for Generating mRNA-Enriched Poly(A)-<br />
Tailed RNA from Total RNA Samples ............................ 18<br />
<strong>EPICENTRE</strong>’s mRNA-ONLY Prokaryotic mRNA Isolation Kit with Poly(A)-Tailing provides<br />
a simple enzymatic process for generating mRNA-enriched poly(A)-tailed RNA from total RNA<br />
samples.<br />
Protein Expression from a Chromosomally-Inserted Transposon. . . . . . 20<br />
Chromosomal protein expression using a transposon-mediated insertion is an alternative for highlevel<br />
expression of proteins in E. coli. Antibiotics or other selective pressures are not required to<br />
maintain the transposon insertion.<br />
Isolate Intron Lariat Loops with RNase R. ........................ 22<br />
Ribonuclease R (RNase R) from E. coli, is a magnesium-dependent 3′→5′ exoribonuclease that<br />
digests essentially all linear RNAs but does not digest lariat or circular RNA structures.<br />
Talk tidbits ................................................ 23<br />
Featuring—<br />
TransforMax EC100 E. coli competent cells<br />
Fast-Link DNA Ligation Kit<br />
On the Cover:<br />
<strong>EPICENTRE</strong> Biotechnologies thanks Dilara Begum for her photo of La Jolla, California. La Jolla (“lah-<br />
HOY-yah”) is a seaside resort community located 15 minutes from downtown San Diego, California.<br />
It borders Pacific Beach to the south and extends north to Torrey Pines State Reserve and Del Mar,<br />
California. La Jolla in Spanish means “The Jewel”, and is home to renowned institutions, such as the<br />
Scripps Institution of Oceanography, the Salk Institute for Biological Studies, and the Stephen Birch<br />
Aquarium & Museum. This is in addition to the University of California, San Diego. Furthermore, La Jolla<br />
is home to many biotechnology and high technology companies.<br />
Hands on the Cover:<br />
Heather Meinholz, <strong>EPICENTRE</strong> Biotechnologies Quality Control<br />
Volume <strong>14</strong> • Number 2<br />
www.EpiBio.com<br />
3
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
ExactStart Full-Length cDNA Library Cloning Kit: A Rapid and<br />
Efficient Method to Synthesize Full-Length cDNA for Cloning and<br />
Accurate Mapping of Transcription Initiation and Polyadenylation Sites<br />
Introduction<br />
The ExactStart Full-Length<br />
cDNA Library Cloning Kit uses a<br />
novel method to clone full-length,<br />
mRNA-derived cDNA, synthesized<br />
from eukaryotic total RNA (FIG<br />
1). The procedure uses Tobacco<br />
Acid Pyrophosphatase to hydrolyze<br />
the phosphoric acid anhydride<br />
bonds in the triphosphate bridge of<br />
the cap structure, thereby releasing<br />
the cap nucleoside and generating<br />
a 5′-monophosphorylated terminus<br />
on the RNA molecule. T4 RNA<br />
ligase is used to add a RNA<br />
acceptor oligo containing a Not I<br />
site to the 5′-end of the mRNA.<br />
A defined 3′ sequence containing<br />
an Asc I site is then added to<br />
the cDNA during the oligo(dT)-<br />
primed reverse transcription of the<br />
mRNA to cDNA. The resulting<br />
full-length, first-strand cDNA is<br />
converted into double-stranded<br />
cDNA (ds cDNA) and amplified by<br />
PCR using primers complementary<br />
to the defined ends of the cDNA.<br />
The amplified ds cDNA is then<br />
cloned using the Not I and Asc I<br />
pre-cut pCDC1 vector included<br />
in the kit. This approach provides<br />
a strong selection for full-length<br />
clones because only 5′-capped and<br />
3′-poly(A)-tailed mRNA is tagged<br />
at both ends; RNA types other<br />
than full-length mRNA will not be<br />
tagged on both ends, and therefore<br />
cannot be amplified or cloned into<br />
the final library.<br />
Ramesh Vaidyanathan, Les M. Hoffman, and John W. Luecke, <strong>EPICENTRE</strong> Biotechnologies<br />
Methods and Results<br />
The ds cDNA was prepared using total RNA<br />
isolated from HeLa cells, and analyzed for the<br />
presence of defined sequences at the 5′- and<br />
3′-terminal ends using β-actin as a candidate<br />
mRNA (FIG 2A). To test whether the β-actin<br />
mRNA is oligo-capped at the 5′-terminal<br />
end, we performed PCR using primers<br />
complementary to the oligo adapter and an<br />
internal primer specific to β-actin. The PCR<br />
results (FIG 2B, Lane 2) show that a product<br />
of expected size is produced, suggesting that<br />
the β-actin mRNA is capped at the 5′-terminal<br />
end. Similar 3′-terminal analysis shows the<br />
FIG 1. Schematic of <strong>EPICENTRE</strong>’s ExactStart Full-Length cDNA Library<br />
Cloning Kit procedure (see text for details).<br />
presence of the tag sequence at the 3′-end of<br />
β-actin mRNA (FIG 2B, Lane 3).<br />
Thirteen clones were then randomly picked<br />
from the cDNA library and analyzed for<br />
insert size by PCR (FIG 3). The results show<br />
a heterogeneous size distribution of inserts<br />
ranging in size from 0.35 kbp to > 2.0 kbp.<br />
Using the ExactStart Full-Length cDNA<br />
Library Cloning Kit as a discovery tool<br />
To show the power and benefits provided<br />
by the ExactStart Kit, cDNA libraries were<br />
prepared from three eukaryotic model<br />
organisms: Saccharomyces cerevisiae,<br />
Schizosaccharomyces pombe, and<br />
Drosophila melanogaster. Clones<br />
from the resulting cDNA libraries<br />
were then sequenced.<br />
Mapping sites of transcription<br />
initiation and polyadenylation<br />
One of the advantages of the<br />
ExactStart Full-Length cDNA<br />
Library Cloning Kit is its ability<br />
to accurately map the sites of<br />
transcription initiation and<br />
polyadenylation in an expressed<br />
gene. The oligo-cap sequence<br />
present at the 5′-termini of<br />
cDNAs defines the mRNA start<br />
site, and the Asc I restriction site<br />
and oligo dA/dT stretch present<br />
at the 3′-termini of cDNA marks<br />
the polyadenylation site. Clones<br />
were analyzed from a size-selected<br />
S. cerevisiae cDNA library prepared<br />
using the ExactStart Kit, and the<br />
sites of transcription initiation and<br />
polyadenylation were mapped. All<br />
of the clones described in FIG 4 are<br />
full-length and contain the 5′- and<br />
3′-terminal sequences.<br />
Novel transcript discovery<br />
Non size-selected cDNA libraries<br />
were made from S. cerevisiae,<br />
S. pombe and D. melanogaster total<br />
RNA; some of the resulting clones<br />
were then sequenced. These RNA<br />
transcripts are almost certainly not<br />
degradation products or incomplete<br />
cDNA transcripts due to the<br />
stringent nature of the ExactStart<br />
Full-Length cDNA Library Cloning<br />
Kit’s tagging and amplification<br />
process. The data show the power of the<br />
ExactStart Kit to find new and potentially<br />
significant RNA transcripts.<br />
Saccharomyces cerevisiae—Several clones<br />
from the S. cerevisiae library were derived from<br />
regions in the genome that are not annotated<br />
as transcriptionally active in the Saccharomyces<br />
Genome Database (http://www.yeastgenome.<br />
org/). Some of these unexpected transcriptional<br />
units, corresponding to antisense, noncoding,<br />
and intergenic regions, correlate well with<br />
data obtained from ChIP-Chip microarray<br />
4<br />
www.EpiBio.com<br />
Volume <strong>14</strong> • Number 2
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
A.<br />
5´-end<br />
B.<br />
3´-end<br />
FIG 2. A. Schematic showing the PCR primer map<br />
positions used for analyzing double stranded HeLa<br />
cDNA for the presence of the defined sequences at the<br />
5′- and 3′-termini (see text for details). B. Agarose gel<br />
analysis of the PCR results; Lane M, mw ladder; Lane 2,<br />
analysis using a 5′-oligo cap and gene specific primer pair;<br />
Lane 3, analysis using a gene specific primer and a primer<br />
for the 3′-tag.<br />
experiments, indicating RNA polymerase<br />
occupancy at or near these sites (David Brow,<br />
University of Wisconsin-Madison, personal<br />
communication). Furthermore, a number<br />
of new RNAs were found by cloning the<br />
“small but not very small” cDNAs (400-500<br />
bp) without selecting for larger, traditional<br />
“gene-sized” cDNAs. The repertory of<br />
“short-ish RNAs” (which we have named<br />
shishRNAs) varies between organisms. If<br />
longer (1-3 kbp) cDNA is selected prior to<br />
cloning, mostly full-length clones of known<br />
transcriptional units predominate in the<br />
library. The small and potentially biologically<br />
relevant transcripts may be missed when larger<br />
cDNA is selected for cloning.<br />
i2280709<br />
FIG 3. PCR analysis of the insert size of thirteen<br />
cDNA clones produced by the ExactStart Full-Length<br />
cDNA Library Cloning Kit. Cloning of a non-size<br />
selected cDNA library can lead to a wide variety of different<br />
sized cDNA clones.<br />
Schizosaccharomyces pombe—The S. pombe<br />
RNA was extracted with <strong>EPICENTRE</strong>’s<br />
MasterPure Yeast RNA Purification Kit. We<br />
found a relatively large number of antisense<br />
transcripts among the S. pombe cDNA clones.<br />
About 6% of the total were examined. Two<br />
of the antisense RNAs were in positions to<br />
interfere with initiation of transcripts from<br />
canonical transcription start sites. Around<br />
60% of the cDNAs encoded ribosomal<br />
proteins, one-third for the small ribosomal<br />
subunit and two-thirds for the large subunit.<br />
Four of the unannotated RNAs contained<br />
no ORFs coding for more than 40 amino<br />
acids. The average S. pombe cDNA length was<br />
470 nt.<br />
Drosophila melanogaster—Fruitfly S2 cell<br />
total RNA was obtained from Ambion. About<br />
55% of the Drosophila cDNA clones encoded<br />
ribosomal proteins, similar to the fraction of<br />
S. pombe clones. A very high percentage of the<br />
cDNAs were apparently full-length. However,<br />
there was an 11:1 ratio of small subunit to<br />
large subunit clones. We have no explanation<br />
for the large imbalance between large and<br />
small subunit protein cDNA clones. The<br />
average fruitfly cDNA length was 535 nt.<br />
Conclusion<br />
The ExactStart Full-Length cDNA Library<br />
Cloning Kit can produce full-length cDNA<br />
libraries from a variety of eukaryotic sources.<br />
These libraries can be size selected to obtain<br />
typical full-length cDNAs, which often show<br />
variations in both 5′- transcriptional start sites<br />
and 3′-polyadenylation addition sites. If size<br />
selection is not performed, the ExactStart<br />
Kit will also clone smaller, unmapped RNA<br />
molecules, which may have important<br />
unknown regulatory effects in the cell.<br />
www.EpiBio.com/exactstart.asp<br />
NEW! ExactStart Full-Length cDNA Library<br />
Cloning Kit<br />
ES0907<br />
10 Reaction Kits<br />
www.EpiBio.com/failsafe.asp<br />
FailSafe PCR PreMix Selection Kit<br />
FS99060 60 Units<br />
Contents: FailSafe PCR Enzyme Mix and all 12<br />
FailSafe PCR 2X PreMixes. Note: Each PreMix<br />
volume has been modified to match the Enzyme<br />
Mix volume.<br />
FailSafe PCR System with PreMix Choice<br />
FS99100 100 Units<br />
Contents: Includes FailSafe PCR Enzyme Mix<br />
and choice of one FailSafe PCR 2X PreMix<br />
(see website above for complete listing).<br />
FailSafe PCR System with PreMix Choice<br />
FS99250 250 Units<br />
Contents: Includes FailSafe PCR Enzyme Mix<br />
and choice of two FailSafe PCR 2X PreMixes<br />
(see website above for complete listing).<br />
FIG 4. Clone analysis from a size-selected S. cerevisiae cDNA library prepared using ExactStart Full-Length<br />
cDNA Library Cloning Kit. Red, 5′- and 3′-terminal sequences; Green, base differences when compared to canonical<br />
sequence; Blue, standard canonical sequence.<br />
These results were presented at the 2007<br />
RNA society meeting and are available online<br />
on the www.EpiBio.com website under<br />
the What’s New/New Publications button<br />
menu (www.EpiBio.com/new_pubs.asp).<br />
Volume <strong>14</strong> • Number 2<br />
www.EpiBio.com<br />
5
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
Fast Real-Time PCR with a Standard qPCR Thermocycler!<br />
Haiying Grunenwald, John W. Luecke, and James Murray, <strong>EPICENTRE</strong> Biotechnologies<br />
Introduction<br />
<strong>EPICENTRE</strong>’s new TAQXpedite GREEN<br />
Real-Time PCR MasterMix Kit generates<br />
reproducible fast real-time PCR (qPCR) results<br />
using a standard real-time thermocycler. The<br />
TAQXpedite GREEN MasterMix Kit achieves<br />
these results by using a highly efficient enzyme<br />
blend of thermostable DNA polymerases with<br />
an optimized 2X MasterMix solution. The<br />
MasterMix contains <strong>EPICENTRE</strong>’s patented<br />
PCR Enhancer (with Betaine),* which<br />
improves PCR amplification, even with the<br />
most difficult templates. 1,2 The data presented<br />
in this short article demonstrate that the easyto-use<br />
TAQXpedite GREEN MasterMix Kit<br />
provides superior results in a fast format.<br />
Benefits of the TAQXpedite GREEN Real-<br />
Time PCR MasterMix Kit include:<br />
• Fast qPCR results with standard qPCR<br />
instrumentation.<br />
• An optimized 2X MasterMix.<br />
• PCR Enhancer (with Betaine).<br />
• High sensitivity and specificity.<br />
Methods and Results<br />
Fast amplification<br />
To evaluate the performance of the<br />
TAQXpedite GREEN Real-Time PCR<br />
MasterMix Kit, a 10-fold serial dilution of<br />
human cDNA ranging from 10 ng to 10 pg<br />
was used as the qPCR template. Each reaction<br />
included: 1X MasterMix, 12.5 pmole of forward<br />
and reverse primers, and the specified amount<br />
of human cDNA. All fast qPCR amplifications<br />
and analyses were performed using a Bio-Rad®<br />
iCycler iQ® thermocycler. The PCR cycling<br />
conditions used were: 35 cycles of 2 seconds<br />
at 92°C and 10 seconds at 70°C for a 210 bp<br />
fragment of the human β-actin gene.<br />
1A<br />
1B<br />
FIG 1. qPCR data for the TAQXpedite GREEN Real-Time PCR MasterMix Kit comparing reactions cycled<br />
immediately after set-up and after incubation for 1 hour at room temperature. Corresponding melt curves indicate<br />
no primer-dimer formation.<br />
TAQXpedite GREEN MasterMix Kit was<br />
tested using experimental conditions where<br />
hot-start is generally recommended. A second<br />
set of qPCR reactions was assembled, and<br />
incubated at room temperature for one<br />
hour before cycling—a process which would<br />
normally permit any primer-dimers or nonspecific<br />
PCR products to form. After cycling,<br />
melt curve and standard curve analyses were<br />
performed to determine the specificity and<br />
efficiency of the PCR reactions.<br />
FIG 1 displays the qPCR quantification curves<br />
(and melt curve analyses) for the TAQXpedite<br />
GREEN MasterMix Kit. Reactions incubated<br />
for 1 hour at room temperature (FIG 1B)<br />
achieved similar qPCR results as those cycled<br />
immediately following set-up (FIG 1A). The<br />
TAQXpedite GREEN MasterMix Kit displayed<br />
excellent R 2 and PCR efficiency data.<br />
The TAQXpedite GREEN MasterMix Kit<br />
produced similar qPCR results, and with<br />
similar high PCR efficiency, in only about<br />
one-third the time as a standard 90-minute<br />
qPCR reaction. In addition, the TAQXpedite<br />
GREEN MasterMix Kit produced excellent<br />
PCR efficiency and correlation factors (R 2 =<br />
0.95–0.99) between replicate amplifications<br />
using fast qPCR conditions.<br />
Excellent PCR efficiency and specificity<br />
In order to simulate conditions which might<br />
be encountered in high-throughput qPCR<br />
using automation, the performance of the<br />
FIG 2. Data showing the qPCR sensitivity, efficiency, and wide dynamic range produced by the TAQXpedite<br />
GREEN Real-Time PCR MasterMix Kit (see text for details). The amplification was linear over the entirety of the<br />
8-fold dilution.<br />
6<br />
www.EpiBio.com<br />
Volume <strong>14</strong> • Number 2
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
Excellent sensitivity and wide<br />
detection range<br />
To examine the sensitivity and detection<br />
range of the kit, a serial dilution of Lambda<br />
DNA ranging from 10 7 to 128 copies was<br />
produced. Each qPCR reaction included:<br />
1X TAQXpedite GREEN Real-Time PCR<br />
MasterMix, 12.5 pmole of forward and<br />
reverse primers, and the appropriate amount<br />
of lambda DNA. Cycling conditions were<br />
30 seconds at 98°C, followed by 35 cycles of<br />
1 second at 92°C and 6 seconds at 70°C.<br />
FIG 2 shows that, in as little as 30 minutes,<br />
the TAQXpedite GREEN MasterMix Kit<br />
produces excellent qPCR sensitivity and<br />
efficiency with a wide dynamic range. The<br />
R 2 values and PCR efficiency indicate that<br />
the TAQXpedite GREEN MasterMix Kit<br />
accurately determines the copy number<br />
present over an extremely wide detection<br />
range, ensuring accurate measurement of even<br />
low abundance transcripts.<br />
Conclusion<br />
The TAQXpedite GREEN Real-Time PCR<br />
MasterMix Kit enables qPCR reactions<br />
to be performed in as little as 30 minutes<br />
using standard qPCR instrumentation. Even<br />
with faster reaction times, results show that<br />
the TAQXpedite GREEN MasterMix Kit<br />
produces excellent values for R 2 and PCR<br />
efficiency while maintaining high sensitivity.<br />
The same excellent qPCR data were obtained<br />
after incubating the TAQXpedite GREEN<br />
reaction mixtures at room temperature for one<br />
hour prior to thermocycling, demonstrating its<br />
applicability in high-throughput applications.<br />
References<br />
1. Henke, W. et al., (1997) Nucl. Acids Res. 25(19),<br />
3957.<br />
2. Abu Al-Soud, W. and Rådström, R. J. Clin.<br />
Microbiol. 38(12), 4463.<br />
www.EpiBio.com/taqxpedite_green.asp<br />
NEW! TAQXpedite GREEN Real-Time PCR<br />
MasterMix Kit<br />
TXG70796 96-25 µl Reactions<br />
TXG707400 400-25 µl Reactions<br />
Contents: 2X MasterMix with Enzyme Blend,<br />
Passive ROX Reference Dye, Stabilizer, and<br />
Nuclease Free Water.<br />
*<br />
Use of Betaine for DNA Polymerase Reactions, including, but not<br />
limited to use for PCR or DNA Sequencing, is covered by, U.S. Patent<br />
No. 6,270,962, European Patent No. 0742838, German Patent No.<br />
DE4411588C1 and other issued or pending applications in the U.S.<br />
and other countries that are either assigned or exclusively licensed to<br />
<strong>EPICENTRE</strong>. These products are accompanied by a limited nonexclusive<br />
license for the purchaser to use the purchased products solely<br />
for life science research. Contact <strong>EPICENTRE</strong> for information on<br />
licenses for uses in diagnostics or other fields.<br />
Easy and Rapid Extraction of Plant DNA<br />
Using the QuickExtract Plant DNA Extraction Solution<br />
Les M. Hoffman and James Murray, <strong>EPICENTRE</strong> Biotechnologies<br />
The QuickExtract Plant Solution<br />
enables the inexpensive processing of one<br />
to hundreds of samples simultaneously<br />
without centrifugation, spin columns,<br />
or the use of toxic organic solvents.<br />
Introduction<br />
Currently, there is no simple effective solution<br />
for high throughput extractions of plant leaf<br />
DNA. Many methods require multiple steps<br />
and often more than one expensive microtiter<br />
dish. Traditional hexadecyltrimethylammonium<br />
bromide (CTAB) based methods<br />
are laborious, and kits based on spin columns<br />
are expensive and not designed for high<br />
throughput. These methods also produce<br />
DNA samples that are likely to contain polyphenolic<br />
compounds and polysaccharides,<br />
which can cause the DNA to be unsuitable<br />
for PCR amplification.<br />
<strong>EPICENTRE</strong>’s new QuickExtract Plant<br />
DNA Extraction Solution is a simple, rapid,<br />
and scalable plant leaf DNA extraction<br />
method for PCR-based applications, including<br />
Random Amplification of Polymorphic DNA<br />
(RAPD) and Short Tandem Repeat (STR)<br />
analyses end-point PCR, or quantitative PCR.<br />
The QuickExtract Plant Solution enables the<br />
FIG 1. RAPD analysis using the QuickExtract Plant<br />
DNA Extraction Kit. One microliter of undiluted<br />
QuickExtract Plant DNA from the leaves of various<br />
species of plants were amplified with the RAPD primer<br />
UBC-888 (5′-BDB(CA) 7<br />
–3′), where B=G/T/C and<br />
D=G/A/T. The PCR products were separated on a 2%<br />
agarose gel and were visualized by staining with SYBR®<br />
Gold. Lane M, 100 bp ladder markers; Lane 1, pepper;<br />
Lane 2, soybean; Lane 3, spelt.<br />
inexpensive processing of one to hundreds<br />
of samples simultaneously without centrifugation,<br />
spin columns, or the use of toxic<br />
organic solvents. It is also an excellent option<br />
for high throughput applications, and is<br />
completely compatible for use with robotic<br />
systems. The 8-minute, 1-tube protocol<br />
requires no freezing, grinding, or bead beating<br />
and yields PCR-ready plant DNA after two<br />
short heating steps.<br />
The QuickExtract Plant Solution has been<br />
successfully used to extract DNA from a wide<br />
range of plant species including corn, soybean,<br />
spelt, pepper, rosemary, grape, spinach,<br />
hopvine and Arabidopsis.<br />
Methods<br />
DNA was extracted from freshly cut 3-5<br />
mm leaf discs of spelt, soybean, pepper, and<br />
spinach. Each leaf disc was placed in an<br />
individual 500 µl tube or microtiter plate, and<br />
100 µl of the QuickExtract Plant Solution was<br />
added to immerse the leaf sample. Care was<br />
taken not to grind or otherwise damage the<br />
leaf tissue as this may release contaminating<br />
polyphenolic compounds. The samples were<br />
then incubated at 65°C for 6 minutes, 98°C<br />
for two minutes, and then placed on ice.<br />
PCR amplification<br />
End-Point PCR—PCR was carried out<br />
using <strong>EPICENTRE</strong>’s FailSafe PCR PreMix<br />
Selection Kit. This kit contains 12 different<br />
PreMixes which enables the rapid optimization<br />
of PCR primers in a single PCR run. One<br />
microliter from each sample of undiluted<br />
QuickExtract Plant DNA was added to each<br />
of the 12 PCR FailSafe PreMixes along with<br />
Continued on Page 8<br />
Volume <strong>14</strong> • Number 2<br />
www.EpiBio.com<br />
7
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
Easy Extraction of Plant DNA - Continued from Page 7<br />
FIG 2. PCR of a single-copy gene using<br />
QuickExtract Plant DNA Extraction Kit template<br />
DNA. One microliter of spinach leaf DNA was obtained<br />
from four different spinach leaves using the QuickExtract<br />
Plant Solution. The PCR products were separated on a<br />
2% agarose gel and visualized by staining with SYBR®<br />
Gold. Lanes 1-4, spinach DNA from four leaves; Lane 5,<br />
100 bp ladder; Lane 6, no DNA negative control.<br />
primers and the FailSafe PCR Enzyme Mix.<br />
The DNA was denatured at 95° for 2 min,<br />
and then PCR amplified for 40 cycles, each<br />
consisting of 95°C for 30 sec, 50°C for 10 sec,<br />
72°C for 45 sec, followed by 72°C for 2 min.<br />
The PCR products were then separated on a<br />
2% agarose gel and visualized by staining with<br />
SYBR® Gold.<br />
Real-Time PCR—Leaf samples were processed<br />
in the same manner as for end-point PCR.<br />
The QuickExtract Plant DNA extracts were<br />
then serially diluted, and, along with negative<br />
control samples, amplified using the FailSafe<br />
GREEN Real-Time PCR System.<br />
Results<br />
DNA was extracted from spelt, soybean<br />
and pepper using the QuickExtract Plant<br />
Solution. DNA from all three samples was<br />
successfully used for RAPD analysis as shown<br />
in FIG 1. PCR on single-copy genes from<br />
four different samples of spinach DNA clearly<br />
demonstrated the high quality and uniformity<br />
of the extracted DNA (FIG 2).<br />
The QuickExtract Plant DNA samples were<br />
also shown to work with real-time PCR<br />
techniques (FIG 3 & 4). The QuickExtract<br />
Plant Solution provided sufficient template<br />
DNA for good PCR results even when the<br />
DNA sample was diluted 1000-fold. With<br />
this efficient DNA extraction, even very small<br />
samples can provide enough template DNA for<br />
hundreds or thousands of qPCR reactions.<br />
Conclusion<br />
The QuickExtract Plant DNA Extraction<br />
Solution represents a rapid and efficient way to<br />
extract PCR-ready plant genomic DNA from<br />
most plant samples. The 8-minute, 1-tube<br />
protocol enables the inexpensive processing<br />
of one to hundreds of samples simultaneously<br />
without centrifugation, spin columns, or<br />
toxic organic solvents. The procedure is fully<br />
compatible with robotic automation, and<br />
produces highly reproducible PCR results.<br />
www.EpiBio.com/quickextract_plant.asp<br />
NEW! QuickExtract Plant DNA<br />
Extraction Solution<br />
QEP70750<br />
50 ml<br />
Bulk solution, sufficient to perform 500 100 µl<br />
extractions.<br />
www.EpiBio.com/failsafe.asp<br />
FailSafe PCR PreMix Selection Kit<br />
FS99060<br />
60 Units<br />
Contents: FailSafe PCR Enzyme Mix and all 12<br />
FailSafe PCR 2X PreMixes.<br />
www.EpiBio.com/failsafegreen.asp<br />
FailSafe GREEN Real-Time<br />
PCR Optimization Kit<br />
FSR0360 96-25 µl Reactions<br />
Contents: FailSafe PCR Enzyme Mix, 12<br />
FailSafe GREEN Real-Time PCR 2X PreMixes,<br />
and Passive Reference Dye.<br />
—— no dilution<br />
—— 1:10<br />
—— 1:100<br />
—— 1:1,000<br />
FIG 3. Real-time PCR using spinach seed<br />
DNA extracted using the QuickExtract<br />
Plant DNA Extraction Kit. The target<br />
amplicon is a 520 bp region of the single-copy<br />
chromosomal HSC70 gene. The results show<br />
clear amplification at a thousand-fold dilution<br />
of the QuickExtract Plant DNA sample.<br />
Negative controls showed no amplification<br />
(data not shown).<br />
—— no dilution<br />
—— 1:10<br />
—— 1:100<br />
—— 1:1,000<br />
FIG 4. Melting point differential plot for<br />
real-time PCR of the spinach leaf HSC70<br />
Gene. The congruence of the melting curves<br />
indicates that the real-time fluorescence was<br />
derived from the expected amplicon.<br />
8<br />
www.EpiBio.com<br />
Volume <strong>14</strong> • Number 2
Evaluation<br />
Kits are now<br />
available!<br />
Detect More Genes Using Your<br />
Illumina ® Expression BeadChips<br />
TargetAmp Nano-g Biotin-aRNA Labeling Kit<br />
for the Illumina ® System<br />
The TargetAmp Nano-g Biotin-aRNA Labeling Kit for the Illumina® System produces Biotin-aRNA (also<br />
called cRNA) for transcription profiling using the Illumina® gene expression system such as the Illumina®<br />
Expression BeadChips. The Biotin-aRNA produced by a TargetAmp Nano-g Kit reaction from 100 ng<br />
of input total RNA has been shown to detect more genes (RNA transcripts) than Biotin-aRNA produced by<br />
another kit using 400 ng of input total RNA.<br />
Benefits<br />
j Produces Biotin-aRNA that detects more genes using<br />
less input total RNA than another kit (Fig 2).<br />
j Produces microgram amounts of Biotin-aRNA with high<br />
signal intensity from as little as 25 ng of input total RNA.<br />
j Easy 6-hour procedure yields Biotin-aRNA ready for<br />
hybridization.<br />
j Linear RNA amplification process preserves the relative<br />
transcript abundance of the sample.<br />
Table 1. The TargetAmp Nano-g Biotin-aRNA Labeling Kit for the<br />
Illumina® System produces microgram amounts of Biotin-aRNA (also called<br />
cRNA) from as little as 25 ng of input total RNA. Results summarize experiments<br />
performed using the HeLa Total RNA control pro vided in the kit.<br />
Input HeLa Control Total RNA<br />
Biotin-aRNA Yield<br />
25 ng 3.1 µg (3100 ng)<br />
50 ng 5.3 µg (5,300 ng)<br />
100 ng 10.8 µg (10,800 ng)<br />
500 ng 75.6 µg (75,600 ng)<br />
FIG 2. Biotin-aRNA (also<br />
called cRNA) produced by a<br />
TargetAmp Nano-g BiotinaRNA<br />
Labeling Kit for the<br />
Illumina® System reaction<br />
detects more genes using less<br />
input total RNA than BiotinaRNA<br />
produced by another<br />
kit. Biotin-aRNA produced<br />
from 100 ng of total RNA using<br />
the TargetAmp Nano-g Kit<br />
and hybridized to the Illumina®<br />
MouseRef-8 Expression BeadChip<br />
detected 1656 transcripts that<br />
were not detected by BiotinaRNA<br />
produced from 400 ng of<br />
total RNA using another kit.<br />
Data courtesy of Dr. Seth Crosby,<br />
Director for Translational<br />
Medicine, Washington University<br />
School of Medicine.<br />
1656 7896 268<br />
Input RNA<br />
100 ng<br />
• <strong>EPICENTRE</strong><br />
TargetAmp Nano-g<br />
Biotin-aRNA Labeling Kit<br />
for the Ilumina ® System<br />
Input RNA<br />
400 ng<br />
• Another<br />
Supplier’s Kit<br />
i2260807<br />
FIG 1. The TargetAmp<br />
Nano-g Biotin-aRNA<br />
Labeling Kit for the Illumina®<br />
System utilizes a linear RNA<br />
amplification and labeling<br />
process that yields purified<br />
Biotin-aRNA in 6 hours.<br />
Ordering Information<br />
TargetAmp Nano-g Biotin-aRNA Labeling Kit for the Illumina ® System<br />
TAN07924<br />
24 Reactions<br />
Find more details using the QuickInfo code in website search box! Go to www.EpiBio.com & use<br />
code: TNDX1
Evaluation<br />
Kits are now<br />
available!<br />
Prepare Biotin-aRNA (cRNA) From as<br />
Little as 5 Cells for Microarray Studies<br />
TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0<br />
The TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 is the only kit that can produce microgram<br />
amounts of Biotin-aRNA (also called cRNA) from as little as 5 cells (about 50 pg of total RNA) for microarray<br />
studies.<br />
Benefits<br />
j The only RNA amplification kit capable of<br />
producing micrograms amounts of Biotin-aRNA<br />
from as little as 5 cells (about 50 pg of input total<br />
RNA).<br />
j Biotin-aRNA produced can be hybridized<br />
to Affymetrix® GeneChip® arrays, Illumina®<br />
Expression BeadChips and other commercial and<br />
spotted arrays that utilize Biotin-labeled target<br />
aRNA.<br />
j Linear RNA amplification process that preserves<br />
the relative transcript abundance of the original<br />
sample.<br />
j Two-day reaction procedure.<br />
Table 1. The TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0<br />
produces high sensitivity Biotin-aRNA (also called cRNA) from minute<br />
samples for microarray studies. Biotin-aRNA was prepared from the specified<br />
amount of mouse liver and skeletal muscle total RNAs using the TargetAmp<br />
2-Round Biotin-aRNA Amplification Kit 3.0 and then hybridized to the<br />
Illumina® MouseRef-8 BeadChip array in duplicates. Data were imported into<br />
BeadStudio (Illumina) for analysis. Genes called “detected” are those with<br />
detection p-value >0.05.<br />
Mouse liver<br />
total RNA<br />
Mouse skeletal<br />
muscle<br />
total RNA<br />
Input total RNA 50 pg 100 pg 50 pg 100 pg<br />
Biotin-aRNA Yield 19.3 µg 35.4 µg 12.1 µg 24.2 µg<br />
Genes (transcripts) detected 7481 8840 7593 8352<br />
Additional TargetAmp aRNA Amplification Kits<br />
that produce Biotin-aRNA, aminoallyl-aRNA, and<br />
unlabeled-aRNA are available. Visit www.EpiBio.com/<br />
targetamp.asp for more information.<br />
Ordering Information<br />
TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0<br />
TAB2R71010<br />
10 Reactions<br />
TAB2R71024<br />
24 Reactions<br />
Find more details using the QuickInfo code in website search box! Go to www.EpiBio.com & use<br />
code: RBDX1
In Vitro Transposomics Tools<br />
Plasmid rescue<br />
The EZ-Tn5 Insertion Kit is ideal<br />
for the recovery and propagation of plasmids or other<br />
circular DNAs that do not contain an E. coli origin of<br />
replication. The EZ-Tn5 transposon will randomly insert<br />
into the plasmid DNA, inserting a kanamycin resistance<br />
marker and the uncommon E. coli plasmid origin R6Kg.<br />
The plasmid can then be recovered and propagated in<br />
either the TransforMax EC100D pir + or pir-116 cells,<br />
depending on the plasmid copy number desired.<br />
Protein Domain & Epitope Mapping<br />
The EZ-Tn5 In-Frame Linker Insertion Kit is<br />
designed to randomly insert 57 base-pair (19 codon)<br />
insertions into any cloned DNA. The insertions can<br />
be translated in all three reading frames, and retain<br />
the original DNA sequence on both sides of the<br />
insertion. This approach is easier and more versatile<br />
than traditional linker scanning mutagenesis.<br />
pir +<br />
pir-116<br />
M 1 2 3 4 5 6 7 8<br />
kb<br />
10 –<br />
Plasmid<br />
DNA<br />
3 –<br />
FIG 1. A plasmid containing an EZ-Tn5<br />
Transposon can be maintained<br />
in TransforMax EC100D pir + cells at ~15 copies<br />
per cell (Lanes 1-4) or in TransforMax EC100D<br />
pir-116 cells at ~250 copies per cell (Lanes 5-8).<br />
i770411<br />
Ordering Information<br />
EZ-Tn5 Insertion Kit<br />
EZI011RK<br />
10 Reactions<br />
TransforMax EC100D pir +<br />
Electrocompetent E. coli<br />
ECP09500 5 x 100 µl<br />
EZ-Tn5 In-Frame Linker Insertion Kit<br />
EZI04KN<br />
10 Reactions<br />
TransforMax EC100D pir-116<br />
Electrocompetent E. coli<br />
EC6P095H 5 x 100 µl<br />
Find more details using the QuickInfo code in website search box! Go to www.EpiBio.com & use<br />
code: EZDX1
In Vitro Transposomics Tools<br />
EZ-Tn5 Promoter<br />
Insertion Kit<br />
The EZ-Tn5 Promoter Insertion Kit is<br />
designed to randomly insert the T7 phage promoter into<br />
any DNA. After insertion, RNA transcripts can be made<br />
using in vitro transcription systems, or in vivo expression<br />
studies can be performed using bacterial strains containing<br />
an inducible T7 RNA polymerase gene.<br />
EZ-Tn5 Transposon<br />
Construction Vectors<br />
Create your own in vitro transposon tools with<br />
our collection of pMOD vectors. These vectors,<br />
which supply the mosaic ends (MEs) needed<br />
for transposition, contain a variety of origins of<br />
replication and antibiotic resistance markers useful<br />
for the construction of custom research tools.<br />
Table 1. Replication origins in EZ-Tn5 Transposon Construction<br />
Vectors.<br />
EZ-Tn5 Transposon<br />
Construction Vectors<br />
ori that is located on<br />
vector outside of the ME<br />
sequences<br />
ori that is located<br />
within the ME<br />
sequences<br />
pMOD-2 colE1 None<br />
pMOD-3 colE1 R6Kgori<br />
pMOD-4 R6Kgori None<br />
pMOD-5 None R6Kgori<br />
pMOD-6* colE1 None<br />
*Contains a Kan R marker inside of the ME<br />
Ordering Information<br />
EZ-Tn5 pMOD Transposon<br />
Construction Vectors:<br />
Ordering Information<br />
-2<br />
MOD0602 20 µg<br />
-3<br />
MOD1503 20 µg<br />
-4<br />
MOD4804 20 µg<br />
-5<br />
MOD4805 20 µg<br />
-6<br />
MOD7906 20 µg<br />
Transposon Construction Vectors include Forward<br />
and Reverse PCR Primers.<br />
EZ-Tn5 Promoter Insertion Kit<br />
EZI03T7 10 Reactions<br />
EZ-Tn5 Transposase<br />
TNP92110 10 Units<br />
Find more details using the QuickInfo code in website search box! Go to www.EpiBio.com & use<br />
code: EZDX1
PCR-ready Plant Genomic DNA<br />
in 8 minutes<br />
QuickExtract Plant DNA Extraction Solution<br />
The QuickExtract Plant DNA Extraction Solution can be used to rapidly and efficiently extract PCR-ready genomic DNA<br />
from most plant samples using a simple 1-tube protocol that takes only 8 minutes. Most leafy plants are acceptable for DNA<br />
extraction using the QuickExtract Plant Solution, including<br />
Arabidopsis, spelt, corn, soybeans, spinach, and pepper leaves<br />
(FIG 1).<br />
The QuickExtract Plant method allows for the inexpensive<br />
processing of one to hundreds of samples simultaneously–<br />
without centrifugation, spin columns or use of any toxic<br />
organic solvent. The procedure is fully compatible with<br />
robotic automation, provides a PCR-ready sample, and<br />
has been proven reproducible (FIG 2). Simply add the<br />
QuickExtract Plant Solution to the sample and perform two<br />
sequential heating steps. A small aliquot of the sample mix is<br />
then used as a template for PCR or qPCR reactions.<br />
Add Plant QuickExtract <br />
DNA Extraction Solution<br />
to sample<br />
Heat at 65ºC for 6 minutes<br />
and 98ºC for 2 minutes<br />
PCR-ready<br />
DNA<br />
i2240707<br />
Applications<br />
j Plant Genomic studies.<br />
j High throughput plant applications.<br />
j GMO testing.<br />
Benefits<br />
j PCR-ready sample.<br />
j No bead beating, freezing or grinding of plant leaf material.<br />
j Nontoxic, inexpensive processing.<br />
j Fast procedure (8 min for average sample).<br />
j No centrifugation.<br />
j Multiple samples can easily be processed simultaneously.<br />
FIG 1. PCR products using QuickExtract Plant DNA<br />
Extraction Solution with different varieties of plant leaves.<br />
40 cycles of RAPD with primer UBC-866 and 1 µl of Plant<br />
QuickExtract DNA preps from leaves with FailSafe PCR<br />
System. Lane M, 100 bp ladder; Lane 1, Pepper; Lane 2,<br />
Soybean and Lane 3, Spelt.<br />
FIG 2. Reproducible PCR Results with<br />
QuickExtract Plant on Arabidopsis<br />
thaliana leaves. Six individual punches<br />
of four Arabidopsis leaves were treated<br />
with QuickExtract Plant Solution.<br />
One microliter of the solution was used<br />
in a 25 µl PCR with the optimized<br />
PreMix of the FailSafe PCR System<br />
and primers specific for the single-copy<br />
HSC70 chromosomal gene. After thirty<br />
five cycles of amplification, aliquots<br />
were electrophoresed in a 2% agarose gel<br />
and the DNA visualized by staining with SYBR Gold. Lanes 1-6, leaf punch<br />
QuickExtract DNAs; Lane 7, no leaf negative control; Lane 8, 100 bp ladder<br />
DNA markers. The expected amplicon is approximately 520 bp.<br />
Ordering Information<br />
QuickExtract Plant DNA Extraction Solution<br />
QEP70750<br />
50 ml<br />
Bulk solution, sufficient to perform 500 100 µl extractions.<br />
Find more details using the QuickInfo code in website search box! Go to www.EpiBio.com & use<br />
code: QPDX1
PCR-ready Genomic DNA<br />
in 3 - 8 minutes<br />
QuickExtract DNA Extraction Solution 1.0<br />
The QuickExtract DNA Extraction Solution 1.0 can be used to rapidly and efficiently extract PCR-ready genomic DNA<br />
from almost any sample type using a simple 1-tube protocol that takes only 3 - 8 minutes, depending on the sample (FIG 1).<br />
Common samples for DNA extraction using QuickExtract Solution include hair follicles, quill-end cells of feathers, tissue culture<br />
cells, buccal cells, and mouse tail snips. It is useful for extracting PCR-ready DNA in high yield for genomic, transgenic, or viral<br />
DNA screening in animals, or for genetic or environmental research and screening in humans or other organisms.<br />
The QuickExtract method allows for the inexpensive processing of one to hundreds of samples simultaneously, without centrifugation,<br />
spin columns or the use of any toxic organic solvent. The method is also compatible with robotic automation (FIG 2).<br />
QuickExtract DNA Extraction Solution is also used in the BuccalAmp DNA Extraction<br />
Kit using a 3-minute single-tube protocol for extraction of PCR-ready DNA from buccal<br />
cells. The method permits the use of samples obtained by non-invasive means to collect<br />
buccal cells with our Catch-All foam swab. The BuccalAmp kit is supplied with our<br />
Catch-All swabs and ready to use 500 µl aliquot tubes of QuickExtract solution.<br />
Tail<br />
Snips<br />
Feathers<br />
Hair<br />
Follicles<br />
Applications<br />
j Simple, rapid extraction of PCRready<br />
DNA.<br />
j Transgenic mouse genotyping.<br />
j Genetic studies.<br />
j Human identity testing.<br />
j Viral or microbial screening.<br />
Benefits<br />
j Nontoxic, inexpensive processing.<br />
j Short procedure.<br />
j No centrifugation.<br />
j High throughput and robotic<br />
compatible.<br />
Bacteria<br />
Add Samples<br />
QuickExtract<br />
Solution<br />
Marker<br />
Human buccal cells<br />
Neoplastic cells (HeLa)<br />
Human hair follicle<br />
Mouse-tail tissue<br />
Bacteria<br />
Transgenic mouse<br />
Heat at 65ºC for 6 minutes<br />
and 98ºC for 2 minutes<br />
M 1 2 3 4 5 6<br />
FIG 1. FailSafe PCR amplifications of genomic DNA extracted<br />
from a variety of tissues or cells. Buccal cells are extracted using<br />
BuccalAmp DNA Extraction Kit, and all others using QuickExtract<br />
DNA Extraction Solution. Lanes 1-3, human β-globin; Lane 4, transgenic<br />
mouse GAPDH; Lane 5, E. coli 16S ribosomal RNA gene; Lane<br />
6, transgenic SV40 T antigen.<br />
PCR-ready<br />
DNA<br />
FIG 2. Procedure for obtaining PCRready<br />
DNA using QuickExtract DNA<br />
Extraction Solution.<br />
Ordering Information<br />
QuickExtract DNA Extraction Solution 1.0<br />
QE09050<br />
50 ml<br />
Bulk solution, sufficient to perform 100 extractions.<br />
BuccalAmp DNA Extraction Kits<br />
BQ0901S<br />
1 Kit (15 Tubes & Swabs)<br />
Catch-All Sample Collection Swabs<br />
QEC091H<br />
100 Swabs<br />
Find more details using the QuickInfo code in website search box! Go to www.EpiBio.com & use<br />
code: QEDX1
FAST End-Point PCR with<br />
a Standard Thermocycler<br />
TAQXpedite PCR System (FAST End-Point)<br />
With a standard thermocycler, the TAQXpedite PCR System has been shown to achieve FAST PCR results by<br />
using enzyme/reagent combinations that improve PCR efficiency. The TAQXpedite PCR System (FAST End-<br />
Point) contains a unique blend of thermostable DNA polymerases that can be used for FAST PCR reactions. The<br />
kit contains the enzyme mix with a carefully optimized 2X Universal MasterMix, and a difficult/long MasterMix<br />
with all four dNTPs, and an optimized MgCl 2<br />
concentration. The MasterMix also contains <strong>EPICENTRE</strong>’s<br />
patented PCR Enhancer (with Betaine), which substantially improves the yield, efficiency, and specificity of<br />
amplification of many target sequences, especially those containing a high GC content or secondary structure.1-⁵<br />
Benefits<br />
j FAST PCR with a standard PCR instrument.<br />
j High fidelity.<br />
j Amplify up to 80% GC content.<br />
j Fast PCR for amplicons longer than 30 kb.<br />
j FAST multiplexing.<br />
j Two convenient MasterMixes for universal and<br />
difficult/long amplifications.<br />
j Fully licensed for PCR research applications.<br />
References<br />
1. Schanke, J.T. and Grunenwald, H.L. (1997) <strong>EPICENTRE</strong> <strong>Forum</strong> 4 (1), 2.<br />
2. Grunenwald, H.L. and Schanke, J.T. (1997) <strong>EPICENTRE</strong> <strong>Forum</strong> 4 (1), 4.<br />
3. Mytelka, D.S. and Chamberlin, M.J. (1996) Nucl. Acids Res. 24, 2774.<br />
4. Henke, W. et al., (1997) Nucl. Acids Res. 25, 3957.<br />
5. Weissensteiner, T. and Lanchbury, J.S. (1996) BioTechniques 21, 1102.<br />
FIG 1. Amplification<br />
using Lambda DNA<br />
as a starting template<br />
for a 539 bp amplicon.<br />
Different starting<br />
concentrations of template<br />
were used in a 16 minute<br />
reaction. Lane 1, kb<br />
ladder; Lane 2, 100 pg;<br />
Lane 3, 10 pg; Lane 4, 1<br />
pg; Lane 5, kb ladder.<br />
FIG 2. A comparison<br />
of TAQXpedite PCR<br />
System, Competitor Q, and<br />
Competitor A, using FAST<br />
PCR to amplify a 4 kb<br />
fragment. Gel image shows<br />
the results starting with 1 ng<br />
Lambda DNA as the template<br />
for the PCR reaction. Lane 1,<br />
MW; Lane 2, TAQXpedite;<br />
Lane 3, Competitor Q’s Fast<br />
Kit; and Lane 4, Competitor<br />
A’s Fast PCR MasterMix.<br />
Ordering Information<br />
TAQXpedite PCR system (FAST End-Point)<br />
TXP78200<br />
200 25-µl Reactions<br />
TXP78001<br />
1,000 25-µl Reactions<br />
Find more details using the QuickInfo code in website search box! Go to www.EpiBio.com & use<br />
code: TEDX1
FAST Real-Time PCR with<br />
a Standard qPCR Thermocycler<br />
TAQXpedite GREEN Real-Time PCR MasterMix Kit<br />
Using a standard Real-Time thermocycler, the TAQXpedite GREEN Real-Time PCR MasterMix Kit is able<br />
to achieve FAST PCR results by using an enzyme/reagent combination to improve the PCR efficiency. The<br />
TAQXpedite GREEN Kit b includes a unique blend of thermostable DNA polymerases with an optimized<br />
2X MasterMix solution containing all four dNTPs, MgCl 2<br />
and SYBR® Green I dye. The MasterMix also<br />
contains <strong>EPICENTRE</strong>’s patented PCR Enhancer (with Betaine a ), which substantially improves the yield<br />
and specificity of amplification of many target sequences, especially those containing a high GC content or<br />
secondary structure. 1-5<br />
Benefits<br />
j FAST reproducible qPCR.<br />
j High sensitivity and specificity.<br />
j Wide dynamic range with excellent PCR efficiencies.<br />
j Convenient 2X MasterMix, plus all reaction compounds<br />
included in kit.<br />
j Fully licensed for qPCR research applications. b<br />
References<br />
1. Schanke, J.T. and Grunenwald, H.L. (1997) <strong>EPICENTRE</strong> <strong>Forum</strong> 4 (1), 2.<br />
2. Grunenwald, H.L. and Schanke, J.T. (1997) <strong>EPICENTRE</strong> <strong>Forum</strong> 4 (1), 4.<br />
3. Mytelka, D.S. and Chamberlin, M.J. (1996) Nucl. Acids Res. 24, 2774.<br />
4. Henke, W. et al., (1997) Nucl. Acids Res. 25, 3957.<br />
5. Weissensteiner, T. and Lanchbury, J.S. (1996) BioTechniques 21, 1102.<br />
FIG 1. Serial dilutions of Lambda DNA from 10 7 to 128 copies were<br />
amplified. Each real-time PCR reaction included 1X MasterMix, 12.5 pmole<br />
each of forward and reverse primers and template. Cycling conditions were<br />
30 seconds at 98°C, followed by 25 cycles of 1 second at 92°C and 6 seconds at<br />
70°C. This serial dilution demonstrates a consistent qPCR reaction and a<br />
reaction efficiency of 103.9%.<br />
Ordering Information<br />
TAQXpedite Ordering Information<br />
GREEN Real-Time PCR MasterMix Kit<br />
TXG70796<br />
96 25-µl Reactions<br />
TXG707400<br />
400 25-µl Reactions<br />
Find more details using the QuickInfo code in website search box! Go to www.EpiBio.com & use<br />
code: TGDX1<br />
SYBR is a registered trademark of Molecular Probes, Inc., Eugene, Oregon. Tween is a registered trademark of ICI Americas Inc., Wilmington, Delaware. TAQXpedite, MasterAmp, MasterPure and FailSafe are<br />
trademarks of <strong>EPICENTRE</strong>, Madison, Wisconsin. SYBR Green I Dye is covered by issued and pending patents owned by Molecular Probes, Inc., Eugene, OR. Products containing SYBR Green Dye are licensed by<br />
<strong>EPICENTRE</strong> for use in enzyme-based nucleic acid amplification procedures, including real-time PCR, solely for life science research.<br />
a<br />
Use of Betaine for DNA Polymerase Reactions, including, but not limited to use for PCR or DNA Sequencing, is covered by, U.S. Patent No. 6,270,962, European Patent No. 0742838, German Patent No.<br />
DE4411588C1 and other issued or pending applications in the U.S. and other countries that are either assigned or exclusively licensed to <strong>EPICENTRE</strong>. These products are accompanied by a limited non-exclusive<br />
license for the purchaser to use the purchased products solely for life science research. Contact <strong>EPICENTRE</strong> for information on licenses for uses in diagnostics or other fields.<br />
b<br />
Covered by U.S. Patent No. 5,436,<strong>14</strong>9 and other patents pending worldwide, owned by Takara Shuzo, Co., Ltd. and licensed to <strong>EPICENTRE</strong>.<br />
<strong>EPICENTRE</strong>’s PCR products are sold under licensing arrangements with F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc., and Applied Biosystems. The products containing a thermostable DNA<br />
polymerase are accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) and RT-PCR for life science research in conjunction with a thermal cycler whose use in the automated performance<br />
of the PCR process is covered by the up-front license fee, either by payment to Applied Biosystems or as purchased, i.e., an authorized thermal cycler. Go to www.EpiBio.com for complete license statements.
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
Questions about RNA capping and in vitro transcription<br />
Q. What is RNA capping<br />
A. Capping is the first step in the maturation<br />
of eukaryotic mRNA. Shortly after the start<br />
of transcription, the 5′-end of the growing<br />
RNA molecule is capped by the addition of<br />
a guanosine monophosphate residue (from<br />
GTP) via a 5′-5′ pyrophosphate linkage. After<br />
addition of the cap nucleotide, a methyl group<br />
is added to the N7 position of the guanine<br />
base producing a Cap 0 structure.<br />
Q. What modifications to in vitro transcribed<br />
RNA are required for efficient translation of<br />
the RNA in vivo<br />
A. For in vitro-transcribed RNA to be<br />
translated efficiently in vivo, the mRNA<br />
requires two things:<br />
• A poly(A)-tail at the 3′-end of the transcript.<br />
• A correctly orientated N7-methylated cap<br />
(Cap 0) at the 5′-end of the molecule.<br />
However, the addition of another methyl<br />
group onto the penultimate nucleotide from<br />
the 5′-end of the mRNA (producing a Cap 1<br />
structure) will further boost translation.<br />
Q. How much better are the translation<br />
efficiencies if I use RNA with a Cap 1<br />
structure instead of a Cap 0 structure<br />
A. It has been reported that methylation<br />
at the 2′-O position of the penultimate<br />
nucleotide of capped RNA (to give a Cap 1<br />
structure) improves translation by 20% to<br />
50% over RNA with a Cap 0 structure.1,2<br />
Q. Which process has better capping<br />
efficiency, <strong>EPICENTRE</strong>’s new mScript<br />
mRNA Production System or cap analog-based<br />
co-transcriptional systems<br />
A. The mScript mRNA Production System<br />
is the first commercially available kit that<br />
produces capped mRNA with nearly 100%<br />
efficiency, and with all of the caps in the<br />
correct orientation.3 Direct incorporation of<br />
cap analog dinucleotides during traditional in<br />
vitro co-transcriptional systems is considerably<br />
less efficient, with capping efficiencies<br />
approaching only 75-80%. In addition, with<br />
some cap analogs, 30% or more of the<br />
caps are incorporated into mRNA in the<br />
wrong orientation, rendering such mRNA<br />
untranslatable.<br />
Q. Why is there such a large improvement<br />
in RNA capping efficiency using the mScript<br />
mRNA Production System when compared to<br />
cap analog-based co-transcriptional reactions<br />
A. Unlike traditional cap analog-based<br />
co-transcriptional reactions that are both costly<br />
and inefficient, the mScript System uses an<br />
enzymatic process to build cap structures onto<br />
the RNA transcripts. Direct incorporation<br />
of cap analog during in vitro transcription is<br />
inconsistent in its capping efficiency and cap<br />
orientation. There is also an inherent trade-off<br />
between capping efficiency and total RNA yield<br />
from the transcription reaction; samples with<br />
high capping efficiencies produce lower overall<br />
RNA yields. The mScript System includes<br />
the Vaccinia virus-derived capping enzyme,<br />
which contains all three enzymatic activities<br />
(mRNA triphosphatase, guanylyltransferase,<br />
and guanine-7-methyltransferase) necessary<br />
to build 5′-Cap 0 structures in vitro, ensuring<br />
nearly 100% capping with 100% proper cap<br />
orientation. Furthermore, the mScript System<br />
contains the Vaccinia 2′-O-Methyltransferase<br />
enzyme, allowing the natural, translationboosting<br />
Cap 1 structure to be built.<br />
Q. Will <strong>EPICENTRE</strong>’s ScriptCap system<br />
work with all in vitro transcribed RNA<br />
A. Yes. ScriptCap Capping Enzyme will work<br />
on any in vitro transcribed RNA provided that<br />
the RNA has a 5′ di- or tri-phosphate.<br />
Q. What is the main benefit of using Anti-<br />
Reverse Cap Analog (ARCA) dinucleotides as<br />
opposed to standard cap analog dinucleotides<br />
for capping of RNA by co-transcriptional<br />
incorporation<br />
Volume <strong>14</strong> • Number 2<br />
A. Standard cap analogs can be incorporated<br />
at the 5′-end of the RNA in both the forward<br />
[m 7 G(5′)ppp(5′)G(pN)...] and the reverse<br />
orientation [G(5′)ppp(5′)m 7 G(pN)...]; RNA<br />
molecules with the cap in the reverse orientation<br />
are not efficiently translated. To overcome this<br />
problem, <strong>EPICENTRE</strong> offers an Anti-Reverse<br />
Cap Analog. This cap analog has a 3′-OCH 3<br />
group on one of the nucleotides, so the ARCA<br />
will be incorporated by the RNA polymerase to<br />
produce RNA transcripts capped exclusively in<br />
the correct orientation.<br />
Q. Which capping system is right for my<br />
needs<br />
A. Generally, mRNA produced using the<br />
mScript mRNA Production System will<br />
outperform the RNA produced using a cap<br />
analog during in vitro transcription. However,<br />
cap analog systems such as the AmpliCap<br />
and MessageMAX kits may have some<br />
advantages for certain applications, see the<br />
table below.<br />
AmpliCap-MAX <br />
T7 and T3 High<br />
Yield Message<br />
Maker Kits<br />
MessageMAX <br />
T7 Capped<br />
Message<br />
Transcription Kit<br />
- ARCA-Capped<br />
mScript <br />
mRNA<br />
Production<br />
System<br />
Reaction time 30 min 30 min 2 hrs<br />
Capping efficiency ~50* ~80% ~100%<br />
Reverse<br />
Yes No No<br />
incorporation of cap<br />
Yield per reaction 60 µg 60 µg 60 µg<br />
Capping of difficultto-cap<br />
Yes Yes Varies<br />
transcripts #<br />
Poly(A) Polymerase No † No † Yes<br />
included<br />
2′-O-Methyltransferase No ‡ No ‡ Yes<br />
included<br />
*Using a typical 4:1 ratio of dinucleotide cap analog to GTP, only about<br />
80% of the transcripts are capped, of which about 60% are in the<br />
correct orientation.<br />
#<br />
These include transcripts with extremely strong 5′ hairpin structures.<br />
Contact Technical Services for more details.<br />
†<br />
The A-Plus Poly(A) Polymerase Tailing Kit can be purchased<br />
separately.<br />
‡<br />
The ScriptCap 2′-O-Methyltransferase can be purchased separately.<br />
References<br />
1. Kuge, H. et al., (1998) Nucl. Acids Res. 26(13),<br />
3208.<br />
2. Meis, R. and Meis, J.E. (2006) <strong>EPICENTRE</strong><br />
<strong>Forum</strong> 13(4), 5.<br />
3. Meis, J.E. and Meis, R. (2007) <strong>EPICENTRE</strong><br />
<strong>Forum</strong> <strong>14</strong>(1), 4.<br />
www.EpiBio.com<br />
17
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
A Simple Enzymatic Process for Generating mRNA-Enriched<br />
Poly(A)-Tailed RNA from Total RNA Samples<br />
Merriann Carey, <strong>EPICENTRE</strong> Biotechnologies<br />
FIG 1. Overview of the mRNA-ONLY Prokaryotic<br />
mRNA Isolation Kit with Poly(A)-Tailing.<br />
Terminator Exonuclease digests the 16S and 23S<br />
rRNAs in prokaryotic RNA to yield an enriched mRNA<br />
preparation. The remaining RNA molecules are then<br />
polyadenylated at the 3′-end using A-Plus Poly(A)<br />
Polymerase in the presence of ATP.<br />
Introduction<br />
Metagenomics has spurred a growing<br />
interest in using environmental mRNA or<br />
transcriptomes to correlate genetic potential<br />
with patterns of microbial activity. However,<br />
progress has been hindered in part by the<br />
difficulties of working with bacterial mRNA.<br />
Bacterial mRNA generally lacks the poly(A)<br />
tail, which makes the isolation and analyses<br />
of eukaryotic messages fairly straightforward.<br />
Moreover, bacterial rRNAs, like their<br />
eukaryotic counterparts, can make up 80%<br />
or more of total RNA, overwhelming samples<br />
with background signals. <strong>EPICENTRE</strong>’s new<br />
mRNA-ONLY Prokaryotic mRNA Isolation<br />
Kit with Poly(A)-Tailing overcomes these<br />
limitations with a simple two-step enzymatic<br />
process that generates mRNA-enriched<br />
poly(A)-tailed RNA from total cellular RNA<br />
samples. The resulting polyadenylated mRNA<br />
can then serve as a template for synthesizing<br />
cDNA for quantitative RT-PCR, cDNA<br />
library construction, and array analysis.<br />
Production of mRNA-enriched<br />
poly(A)-tailed RNA<br />
The two-step enzymatic process provided<br />
by the mRNA-ONLY Prokaryotic mRNA<br />
Isolation Kit with Poly(A)-Tailing is shown<br />
in FIG 1. <strong>EPICENTRE</strong>’s Terminator<br />
5′-Phosphate-Dependent Exonuclease* is<br />
used to digest ribosomal RNAs having a<br />
5′-monophosphate. This processive 5′–3′<br />
exonuclease does not digest RNA having a<br />
5′-triphosphate, a 5′-cap structure (present on<br />
most eukaryotic mRNAs), or a 5′-hydroxyl<br />
group. Consequently, Terminator Exonuclease<br />
can be used to isolate prokaryotic mRNA with<br />
minimal carryover of 16S and 23S rRNA. 1<br />
The remaining RNA molecules are then<br />
polyadenylated at the 3′-end using A-Plus<br />
Poly(A) Polymerase in the presence of ATP.<br />
First-Strand cDNA Synthesis<br />
As a simple test case, mRNA-enriched<br />
poly(A)-tailed E. coli RNA, prepared using<br />
the mRNA-ONLY Prokaryotic mRNA<br />
Isolation Kit with Poly(A)-Tailing, was reverse<br />
transcribed using an oligo(dT) primer and<br />
MMLV Reverse Transcriptase (MMLV RT).<br />
The resulting cDNA was used for real-time<br />
PCR (qPCR) without additional treatment<br />
or purification. qPCR was performed using<br />
the TAQurate GREEN Real-Time PCR<br />
MasterMix. a Control reactions were also<br />
performed under the same conditions except<br />
that MMLV RT was omitted.<br />
FIG 2. Analysis of mRNA-enriched poly(A)-tailed<br />
E. coli RNA by real-time RT-PCR. Each amplification<br />
plot is color coded and the corresponding gene designations<br />
are listed in Table 1.<br />
Target ETCN C T<br />
fliC 51 17.2<br />
rplK 51 17.9<br />
pal 18 18.1<br />
cyoA 16 20.6<br />
ilvE 4 22.0<br />
basR 2 22.4<br />
fecD 0.3 25.9<br />
yafL 0.1 27.0<br />
Table 1. Comparison of<br />
Estimated Transcription<br />
Copy Number (ETCN)<br />
and Cycle Threshold (C T<br />
)<br />
Values.<br />
As shown in FIG 2 and Table 1, the average<br />
cycle threshold (C T<br />
) value for each of the<br />
eight targets tested correlates well with the<br />
estimated transcription copy number (ETCN)<br />
derived from E. coli microarray data. 2 Thus,<br />
a highly expressed gene such as fliC with<br />
an ETCN of 51 has a correspondingly high<br />
real-time RT-PCR signal (low C T<br />
value). Even<br />
the 3-fold difference in ETCN levels for the<br />
rare sequences fecD and yafL was represented<br />
by reproducible differences in C T<br />
values.<br />
Preservation of relative mRNA<br />
abundance levels<br />
To evaluate the effect of Terminator<br />
Exonuclease and A-Plus Poly(A) Polymerase<br />
on the relative abundance of the eight mRNA<br />
transcripts listed in Table 1, real-time RT-PCR<br />
was performed before and after treatment with<br />
these two enzymes. The “untreated” cDNA<br />
was produced from total E. coli RNA using<br />
MMLV RT and random nonamer primers.<br />
The “treated” cDNA was produced as described<br />
in the previous section. The results shown in<br />
FIG 3 are expressed as the difference in C T<br />
values (∆C T<br />
) between the target message and<br />
the normalizer (pal) for untreated and treated<br />
samples. The high correlation coefficient<br />
of R 2 =0.935 demonstrates that the cDNA<br />
produced from the mRNA-enriched poly(A)-<br />
tailed RNA conserved the relative abundance<br />
of different RNA species.<br />
Amplification of environmental mRNA<br />
To validate the efficacy of Terminator<br />
Exonuclease and A-Plus Poly(A) Polymerase<br />
in the analysis of environmental RNA, a<br />
fresh sample of dog feces, known to be rich<br />
in bacteria, was used. Initially, DNA isolated<br />
from dog feces (using the MasterPure DNA<br />
Purification Kit) was screened by end-point<br />
PCR with the same set of primers used to<br />
amplify the E. coli genes listed in Table 1.<br />
Four of the eight PCR reactions (cyoA, pal,<br />
rplK, and yafL) yielded amplicons of the<br />
expected size (data not shown).<br />
Total RNA isolated from the same fecal<br />
sample as the genomic DNA, was then treated<br />
sequentially with Terminator Exonuclease and<br />
A-Plus Poly(A) Polymerase (FIG 4). Since the<br />
amount of total RNA from environmental<br />
samples is likely to be small, the poly(A)-tailed<br />
RNA produced by the Terminator Exonuclease<br />
and A-Plus Poly(A) Polymerase treatment<br />
was amplified using the MessageBOOSTER<br />
cDNA Synthesis Kit for qPCR b before further<br />
Relative Expression (∆C T )<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
-2<br />
yafL fecD basR ilvE cyoA rplK fliC<br />
RNA Target<br />
Untreated<br />
Terminator Exonuclease +<br />
A-Plus Poly(A) Polymerase<br />
FIG 3. Treatment of E. coli RNA with Terminator<br />
Exonuclease and A-Plus Poly(A) Polymerase maintains<br />
relative mRNA abundance levels as determined<br />
by real-time RT-PCR.<br />
c1930705<br />
18<br />
www.EpiBio.com<br />
Volume <strong>14</strong> • Number 2
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
FIG 4. Denaturing agarose<br />
gel analysis of total fecal<br />
RNA before (-) and after (+)<br />
digestion with Terminator<br />
Exonuclease. The mRNAenriched<br />
sample was then<br />
polyadenylated with A-Plus<br />
Polymerase (data not shown).<br />
analysis. MessageBOOSTER reactions<br />
utilize a linear RNA amplification process<br />
that produces large amounts of anti-sense<br />
RNA (aRNA, also called cRNA) from poly(A)<br />
RNA, and then converts the aRNA to cDNA<br />
for qPCR. 3<br />
One hundred picograms of mRNA-enriched<br />
poly(A)-tailed fecal RNA was used in a<br />
standard MessageBOOSTER reaction, and<br />
5 to 15% of the resulting cDNA was then<br />
used for real-time RT-PCR. As shown in<br />
FIG 5A, two of the four targets tested (pal<br />
and yafL) yielded satisfactory qPCR signals.<br />
The specificity and size of the PCR products<br />
were verified by melting curve analysis and<br />
gel electrophoresis (FIG 5, B and C). qPCR<br />
of cDNA produced from 100 pg of enriched<br />
and tailed RNA, without the benefit of a<br />
MessageBOOSTER reaction, failed to detect<br />
any of these four targets (data not shown).<br />
Panel A<br />
Conclusion<br />
Global analysis of microbial transcripts in<br />
environmental samples typically relies on the<br />
conversion of RNA to cDNA via random<br />
priming. Random priming coupled with the<br />
abundance of rRNA can overwhelm low<br />
level transcripts so that their subsequent<br />
amplification may not be quantitative. The<br />
specificity of oligo(dT) priming is therefore<br />
often cited as the best method to use when<br />
a faithful cDNA representation of the<br />
mRNA pool is desired. 4 Moreover, oligo(dT)<br />
priming is the most appropriate choice when<br />
amplification of mRNAs from limited RNA<br />
samples is desired.<br />
The results shown here demonstrate that the<br />
mRNA-ONLY Prokaryotic mRNA Isolation<br />
Kit with Poly(A)-Tailing provides a simple<br />
enzymatic process for generating mRNAenriched<br />
poly(A)-tailed RNA from total RNA<br />
samples. The resulting polyadenylated mRNA<br />
lacks the confounding effects of rRNA and<br />
makes an ideal template for cDNA synthesis<br />
using an oligo(dT) primer.<br />
References<br />
1. Meis, J. E. and Pease, J. (2006) <strong>EPICENTRE</strong><br />
<strong>Forum</strong> 13(1), 22.<br />
2. Liu, M. et al., (2005) J. Biol. Chem. 280(16),<br />
15921.<br />
3. Grunenwald, H. et al., (2006) <strong>EPICENTRE</strong> <strong>Forum</strong><br />
13(1), 7.<br />
4. Bustin, S.A. et al., (2005) J. of Mol. Endocrinol<br />
34(3), 597.<br />
*All products used in this article are from <strong>EPICENTRE</strong><br />
Biotechnologies unless otherwise stated.<br />
Panel B<br />
www.EpiBio.com/mrnaonly.asp<br />
mRNA-ONLY Prokaryotic mRNA Isolation Kit<br />
with Poly(A)-Tailing<br />
MOT60510<br />
10 Reactions<br />
Contents: Terminator 5′-Phosphate-Dependent<br />
Exonuclease, mRNA-ONLY 10X Reaction Buffer,<br />
RNase Inhibitor, mRNA-ONLY Stop Solution,<br />
5M LiCl Solution, A-Plus Poly(A) Polymerase,<br />
10X A-Plus Reaction Buffer, 10 mM ATP, and<br />
RNase-Free Water.<br />
www.EpiBio.com/mmlv.asp<br />
MMLV Reverse Transcriptase<br />
M6125H 10 U/µl 2,500 Units<br />
M6110K 10 U/µl 10,000 Units<br />
M4425H 50 U/µl 2,500 Units<br />
M4410K 50 U/µl 10,000 Units<br />
Includes 10X Reaction Buffer and DTT.<br />
www.EpiBio.com/taqurate.asp<br />
TAQurate GREEN Real-Time PCR MasterMix<br />
TM049096 96 25-µl Reactions<br />
TM046400 400 25-µl Reactions<br />
Contents: TAQurate Real-Time PCR Enzyme<br />
Blend, TAQurate GREEN Real-Time 2X PCR<br />
MasterMix, Passive Reference Dye, and Stabilizer.<br />
www.EpiBio.com/masterpure_complete.asp<br />
MasterPure DNA Purification Kit<br />
(for isolating TNA or DNA)<br />
MCD85201 200 Purifications<br />
Contents: Red Cell Lysis Solution, Tissue and Cell<br />
Lysis Solution, MPC Protein Precipitation Reagent,<br />
2X T&C Lysis Solution, TE Buffer, RNase A, and<br />
Proteinase K.<br />
www.EpiBio.com/messagebooster.asp<br />
MessageBOOSTER cDNA Synthesis Kit<br />
for qPCR<br />
MB060110<br />
10 Reactions<br />
MB060124<br />
24 Reactions<br />
Panel C<br />
cyoA pal<br />
M 1 2 3 4<br />
rplK yafL<br />
M 5 6 7 8<br />
a<br />
<strong>EPICENTRE</strong>’s PCR products are sold under licensing arrangements<br />
with F. Hoffmann-La Roche Ltd., Roche Molecular Systems, Inc.,<br />
and Applied Biosystems. The products containing a thermostable<br />
DNA polymerase are accompanied by a limited license to use it in<br />
the Polymerase Chain Reaction (PCR) and RT-PCR for life science<br />
research in conjunction with a thermal cycler whose use in the automated<br />
performance of the PCR process is covered by the up-front<br />
license fee, either by payment to Applied Biosystems or as purchased,<br />
i.e., an authorized thermal cycler.<br />
b<br />
MessageBOOSTER is a trademark of <strong>EPICENTRE</strong> Biotechnologies,<br />
Madison, WI. This product is covered by intellectual property licensed<br />
to <strong>EPICENTRE</strong> Technologies Corporation from Johnson & Johnson<br />
Pharmaceutical Research & Development, L.L.C. This product is<br />
covered by U.S. Patents licensed exclusively to Incyte Corporation and<br />
sublicensed to <strong>EPICENTRE</strong> Technologies Corporation. See www.<br />
EpiBio.com website for complete license statements.<br />
FIG 5. Real-time RT-PCR analysis of mRNA-enriched poly(A)-tailed fecal RNA. Panel A, qPCR graphs for pal and<br />
yafL with no template controls; Panel B, melt-curve analysis; Panel C, Gel electrophoresis of RT-PCR products from<br />
E. coli (odd lanes) and fecal (even lanes) samples.<br />
Volume <strong>14</strong> • Number 2<br />
www.EpiBio.com<br />
19
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
Protein Expression from a Chromosomally-Inserted Transposon<br />
Darin J. Haskins, <strong>EPICENTRE</strong> Biotechnologies<br />
20<br />
Introduction<br />
The expression of proteins in microorganisms<br />
for research or commercial applications is<br />
accomplished almost exclusively using<br />
plasmid-based expression systems. An example<br />
is the bacteriophage T7 expression system<br />
pioneered by Dr. William Studier,1 which<br />
is used by many researchers to obtain a<br />
high-level expression of cloned proteins in E.<br />
coli. This system utilizes a plasmid in which<br />
the gene of interest is cloned downstream of<br />
the phage T7 gene 10 promoter and a host<br />
strain that is capable of expressing T7 RNA<br />
polymerase upon induction.<br />
Although plasmid-based expression systems<br />
can be very useful, they are not without<br />
problems. Plasmids can place a large metabolic<br />
burden on host cells. Therefore, antibiotics<br />
may be required in the growth medium in<br />
order to avoid loss of the plasmid during<br />
cultivation.2 Furthermore, employment of<br />
a strong plasmid selection method can kill<br />
cells that lose the plasmid, leading to an<br />
accumulation of non-viable and lysed cell<br />
matter, which complicates purification.3<br />
Overexpression of the cloned gene or other<br />
genes located on the plasmid can also lead to<br />
lower plasmid segregation stability.2<br />
The standard practice of using antibiotic<br />
selection for plasmid maintenance also limits<br />
which plasmids and antibiotic resistance genes<br />
can be used for production of pharmaceutical<br />
grade proteins. This is because regulatory<br />
standards do not allow use of certain antibiotics<br />
because of the potential for patient allergic<br />
reactions to low levels of residual antibiotics.<br />
An expression transposon, stably integrated<br />
into the chromosome of a microorganism,<br />
can alleviate many problems related to<br />
protein expression. The transposon can<br />
be inserted into the host chromosome using<br />
an EZ-Tn5 Transposome complex<br />
(<strong>EPICENTRE</strong> Biotechnologies). In this<br />
method, the transposase is pre-bound in vitro<br />
to the 19-base mosaic ends (i.e., transposase<br />
recognition sequences) of an “artificial”<br />
transposon in the absence of Mg ++ , and then<br />
electroporated into the microbe. Once inside<br />
the cell, this EZ-Tn5 Transposome is activated<br />
by Mg ++ in the host’s cellular environment,<br />
where the Transposome efficiently and<br />
randomly inserts its transposon DNA into<br />
the genomic DNA of the host cell. The<br />
transposon is called “artificial” because it lacks<br />
www.EpiBio.com<br />
FIG 1. T7 expression cassettes were engineered into the EZ-Tn5 pMOD-6 Transposon<br />
Construction Vector in the configuration shown above.<br />
a gene that encodes the transposase. Therefore,<br />
the transposon is completely stable and cannot<br />
excise or reinsert itself into a new position<br />
on the chromosome.4 Antibiotics or other<br />
selective pressures are not required to maintain<br />
the transposon insertion, and if desired, the<br />
antibiotic resistance marker used to select for<br />
the initial insertion can be removed by standard<br />
genetic engineering methods.<br />
Any DNA can be used to make an<br />
EZ-Tn5 transposon simply by adding<br />
transposase recognition sequences to both<br />
ends. Here, we report construction and use<br />
of EZ-Tn5 Transposomes for expression<br />
of two different proteins from the E. coli<br />
chromosome.<br />
Methods and Results<br />
Protein over-expression<br />
T7 expression cassettes were engineered into<br />
the new EZ-Tn5 pMOD-6 Transposon Construction Vector in<br />
the configuration seen in FIG 1. Briefly,<br />
transcription of the gene to be expressed is<br />
driven by a T7 promoter located upstream<br />
of the open reading frame. The kanamycin<br />
resistance gene is included in the construct to<br />
provide a marker for the initial insertion event<br />
of the Transposome DNA into the E. coli<br />
chromosome. It is not necessary to utilize this<br />
marker in subsequent culturing, and it can be<br />
removed post-insertion if desired.<br />
The Transposome complex was created by<br />
cutting the protein expression cassette from<br />
the pMOD vector using the Pvu II restriction<br />
sites outside of the mosaic ends. Alternatively,<br />
transposon DNA can be generated by<br />
standard PCR as described in <strong>EPICENTRE</strong>’s<br />
product literature (www.EpiBio.com). The<br />
transposon DNA was then incubated with<br />
EZ-Tn5 transposase in the absence of Mg ++ ,<br />
allowing the stable Transposome complex<br />
to form.<br />
Volume <strong>14</strong> • Number 2<br />
The Transposome was then electroporated<br />
into the widely-used protein expression strain<br />
BL21(DE3). As described above, intracellular<br />
Mg ++ then activates the transposase, allowing<br />
DNA insertion to occur. Chromosomal<br />
insertions were selected for by plating on<br />
kanamycin-containing LB solid medium, and<br />
confirmed by screening for the lack of a<br />
circular plasmid, potentially due to uncut<br />
pMOD vector background. The presence of<br />
each insertion on the chromosome can be<br />
confirmed by purifying total cellular DNA<br />
using the MasterPure DNA Purification Kit<br />
(<strong>EPICENTRE</strong>) and performing sequencing<br />
reactions using primers that anneal to the ends<br />
of the inserted EZ-Tn5 transposon.<br />
Two different proteins were expressed in this<br />
manner to demonstrate the effectiveness of<br />
this approach. Three random chromosomal<br />
insertion clones were selected for each protein<br />
and assayed for the level of protein production<br />
as compared with the same proteins cloned<br />
into a pET T7 expression plasmid (Novagen).<br />
Protein yields were measured by lysing<br />
equivalent amounts of total cell mass and<br />
running them on an SDS-PAGE gel. The<br />
band intensities of the induced protein of<br />
interest were visually compared.<br />
As seen in FIG 2, the expression of Protein X<br />
when using a chromosomally inserted EZ-Tn5<br />
transposon was only very slightly lower than<br />
the expression achieved when using a pET<br />
plasmid. As for Protein Y, expression levels<br />
were very similar in E. coli cells, whether<br />
the protein was expressed using an EZ-Tn5<br />
transposon or from a pET plasmid (FIG 3).<br />
Conclusion<br />
Chromosomal protein expression using<br />
transposon-mediated insertion is a viable<br />
alternative for high-level expression of proteins<br />
in E. coli. EZ-Tn5 transposon-based expression<br />
resulted in production of similar amounts
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
www.EpiBio.com/pmod.asp<br />
FIG 2. Expression of protein<br />
X from a pET plasmid or a<br />
chromosomally-inserted EZ-Tn5<br />
transposon. Equivalent amounts of<br />
un-induced(-) and induced(+) total<br />
cell lysates based on OD 600<br />
values<br />
were separated on a 10% SDS-<br />
PAGE. Lanes 1-2, pET expressed<br />
protein X; Lane 3, 250ng purified<br />
protein X standard; Lanes 4-9,<br />
chromosomal EZ-Tn5 transposon<br />
protein X expression clones.<br />
EZ-Tn5 pMOD-2 Transposon<br />
Construction Vector<br />
MOD0602 20 µg<br />
EZ-Tn5 pMOD-3<br />
Transposon Construction Vector<br />
MOD1503 20 µg<br />
EZ-Tn5 pMOD-4 Transposon<br />
Construction Vector<br />
MOD4804 20 µg<br />
EZ-Tn5 pMOD-5<br />
Transposon Construction Vector<br />
MOD4805 20 µg<br />
NEW! EZ-Tn5 pMOD-6<br />
Transposon Construction Vector<br />
MOD7906 20 µg<br />
FIG 3: Expression of protein<br />
Y from a pET plasmid or a<br />
chromosomally-inserted EZ-Tn5<br />
transposon. Equivalent amount of<br />
un-induced(-) and induced(+) total<br />
cell lysates based on OD 600<br />
values<br />
were separated on a 10% SDS-<br />
PAGE. Lanes 1-2, pET expressed<br />
protein Y; Lane 3, 250ng purified<br />
protein Y standard; Lanes 4-9,<br />
chromosomal EZ-Tn5 transposon<br />
protein Y expression clones.<br />
www.EpiBio.com/transposase.asp<br />
EZ-Tn5 Transposase<br />
TNP92110 10 U<br />
www.EpiBio.com/ec100d_pir.asp<br />
TransforMax EC100D pir +<br />
Electrocompetent E. coli<br />
ECP09500 5 x 100 µl<br />
Includes control vector containing an R6Kγori.<br />
of two different proteins as a commonly<br />
used plasmid expression system. EZ-Tn5<br />
Transposomes provide researchers with an<br />
easy-to-use, well-characterized tool for creating<br />
these chromosomal expression strains.<br />
Stable chromosomal insertion of heterologous<br />
genes in EZ-Tn5 transposons enable expression<br />
of high levels of proteins without use of<br />
antibiotics, which is desirable in all production<br />
situations, but especially when operating under<br />
pharmaceutical GMP conditions. The ability<br />
to stably maintain chromosomal expression<br />
strains without antibiotic selection additionally<br />
facilitates validation and verification of GMP<br />
production strains. Chromosomal expression<br />
of transposon-inserted genes also helps<br />
eliminate other problematic issues associated<br />
with plasmid based expression systems such<br />
as plasmid loss, and accumulation of excessive<br />
cellular debris from non-productive cells.<br />
Thus, chromosomal protein expression using<br />
the EZ-Tn5 system can increase recombinant<br />
protein yields, lower the costs associated<br />
with culture media, and simplify the protein<br />
purification process.<br />
In addition to its uses for manufacturing<br />
proteins, the EZ-Tn5 Transposome expression<br />
technology can be used to genetically engineer<br />
bacterial strains to express non-native proteins<br />
for specific tasks, such as bio-remediation<br />
or for conversion of biomass into useful<br />
materials, and for production of biofuels.<br />
<strong>EPICENTRE</strong>’s EZ-Tn5 Transposome<br />
expression technology is a powerful tool for<br />
researchers and bioengineers who wish to<br />
“customize” microbes or express proteins at<br />
high level for downstream applications.<br />
References<br />
1. Studier, F. W. and Moffatt, B. A. (1986) J. Mol.<br />
Biol. 189(1), 113.<br />
2. Friehs, K. (2004) Adv. Biochem. Eng. Biotechnol.<br />
86, 47.<br />
3. Jahic, M. et al., (2003) Microb. Cell Fact. 2(1), 6.<br />
4. Goryshin, I. W. et al., (2000) Nat. Biotechnol.<br />
18(1), 97.<br />
TransforMax EC100D pir-116<br />
Electrocompetent E. coli<br />
EC6P095H 5 x 100 µl<br />
Includes control vector containing an R6Kγori.<br />
www.EpiBio.com/r6kgori_kan2_transposome.asp<br />
EZ-Tn5 Tnp<br />
Transposome Kit<br />
TSM08KR 10 Reactions<br />
www.EpiBio.com/kan2_tnp_transposome.asp<br />
EZ-Tn5 Tnp Transposome Kit<br />
TSM99K2 10 Reactions<br />
EZ-Tn5 Tnp Transposome Kit<br />
TSM99D1 10 Reactions<br />
Each kit contains a ready-to-use Transposome<br />
complex with either a kanamycin resistance marker<br />
or dihydrofolate reductase gene (for selection with<br />
trimethoprim) and forward and reverse primers for<br />
sequencing.<br />
This technology is covered by patents exclusively licensed to<br />
<strong>EPICENTRE</strong>. Nonprofit organizations receive a research license<br />
by purchase of EZ-Tn5 or HyperMu transposon tools (visit<br />
http://www.EpiBio.com/terms.asp#patents for detailed licensing<br />
information). For-profit organizations are allowed to use in vivo<br />
transposition technology and tools solely for evaluation purposes<br />
for up to three months, after which a license will be required for<br />
the continued use of the patented technology, tools and derivatives.<br />
For questions and inquiries please contact Stefan Koehler at<br />
stefan.koehler@EpiBio.com.<br />
Volume <strong>14</strong> • Number 2<br />
www.EpiBio.com<br />
21
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
Isolate Intron Lariat Loops with RNase R<br />
Les M. Hoffman and James Murray, <strong>EPICENTRE</strong> Biotechnologies<br />
Precursor mRNA or pre-mRNA is an<br />
incompletely processed single-strand of<br />
mRNA synthesized from a DNA template<br />
in the nucleus of a cell. The processing of<br />
pre-mRNA by splicing has long been of interest<br />
to researchers studying gene regulation. RNA<br />
splicing is a spliceosome mediated event that<br />
removes non-coding intervening sequences<br />
(introns) from pre-mRNA and releases them<br />
in the form of branched lariat structures<br />
that are later degraded. Alternative splicing<br />
locations of introns can have a dramatic<br />
effect on the resulting translation product.<br />
<strong>EPICENTRE</strong>’s Ribonuclease R (RNase R)<br />
can be used to study such splicing events by<br />
digesting mRNA from this complex mixture<br />
leaving the intron lariat loops intact (FIG 1).<br />
Ribonuclease R from E. coli, is a magnesiumdependent<br />
3′→5′ exoribonuclease that digests<br />
essentially all linear RNAs but not lariat or<br />
circular RNA structures.1,2 It also digests<br />
double-stranded RNA with 3′-overhangs<br />
shorter than seven nucleotides.2 Most cellular<br />
RNAs will be digested completely by RNase<br />
R, with the exception of tRNAs, 5S RNA,<br />
and intron lariats. The 3′-tails of lariats will<br />
be trimmed by RNase R to the branch<br />
point nucleotide, where there is a 2′,5′-<br />
phosphodiester linkage. Lariats produced<br />
during pre-mRNA splicing of intron regions<br />
can be isolated from a mixture of total RNA<br />
by digestion with RNase R. <strong>EPICENTRE</strong>’s<br />
ArrayPure Nano-scale RNA Purification<br />
Kit, MasterPure RNA Purification Kit, and<br />
MasterPure Yeast RNA Purification Kits<br />
are ideal products for obtaining total RNA<br />
preparations.<br />
RNA isolated using RNase R can be employed<br />
as a template to produce labeled cDNA with<br />
random primers. Intronic cDNA molecules can<br />
be used as a target for microarrays containing<br />
Pre-mRNA<br />
mRNA<br />
Ribonuclease R (RNase R)<br />
Exon 1 Intron Exon 2<br />
Intron<br />
Exon 1 Exon 2<br />
Exon 1 Exon 2<br />
Intron<br />
Intron<br />
RNase R Digestion<br />
Lariat Only Remains<br />
FIG 1. Introns are spliced out from pre-mRNA and<br />
released in the form of a branched lariat structure.<br />
RNase R degrades linear RNAs while preserving the loop<br />
portion of a lariat RNA.<br />
potential intron sequences, or for tiling arrays<br />
containing overlapping regions of complete<br />
chromosomes or genomes. The cDNA<br />
produced will not be a linear representation of<br />
the intron, as the lariat has no starting point<br />
of origin, but the sequences contained in it<br />
will be intron-derived. RNase R is provided<br />
in a 250 U size (20 U/µl or 1 µg/µl) and is<br />
supplied with a 10X Reaction Buffer.<br />
Applications<br />
• Alternative mRNA splicing studies.<br />
• Gene expression studies.<br />
• Intron cDNA production.<br />
• Intronic screening of cDNA libraries.<br />
i2220706<br />
References<br />
1. Suzuki, H. et al., (2006) Nucleic Acids Res. 34(8),<br />
e63.<br />
2. Vincent, H. A. and Deutscher, M.P.(2006) J. Biol.<br />
Chem. 281(40), 29769.<br />
www.EpiBio.com/rnaser.asp<br />
NEW! Ribonuclease R (RNase R)<br />
RNR07250<br />
250 Units<br />
www.EpiBio.com/arraypure.asp<br />
ArrayPure Nano-scale RNA Purification Kit<br />
MPS04050<br />
50 Purifications<br />
www.EpiBio.com/masterpure_complete.asp<br />
MasterPure RNA Purification Kit<br />
(for isolating RNA only)<br />
MCR85102 100 Purifications<br />
Contents: Red Cell Lysis Solution, Tissue and Cell<br />
Lysis Solution, MPC Protein Precipitation Reagent,<br />
2X T & C Lysis Solution, TE Buffer, RNase-Free<br />
DNase I, Proteinase K, and 1X DNase Buffer.<br />
www.EpiBio.com/masterpure_yrna.asp<br />
MasterPure Yeast RNA Purification Kit<br />
MPY03010 10 Reactions<br />
MPY03100 100 Reactions<br />
Contents: Extraction Reagent for RNA, MPC<br />
Protein Precipitation Reagent, TE Buffer (in 100<br />
rxn kit only), Proteinase K, RNase-Free DNase I,<br />
10X DNase Buffer, and 2X T & C Lysis Solution.<br />
Kits and Reagents for<br />
In Vitro Transcription<br />
& RNA Research<br />
Kits and Reagents for<br />
Nucleic Acid Extraction<br />
& Purification Research<br />
Ask for the latest literature available—<strong>EPICENTRE</strong> SPOTLIGHT Brochures<br />
22<br />
www.EpiBio.com<br />
Volume <strong>14</strong> • Number 2
<strong>EPICENTRE</strong>® Biotechnologies <strong>Forum</strong><br />
Talktidbits<br />
TransforMax EC100 E. Coli Competent Cells<br />
High Efficiency Electrocompetent & Chemically Competent E. Coli<br />
The highly versatile TransforMax<br />
EC100 E. coli competent cells are ideal<br />
for most cloning applications. The cells<br />
provide very high transformation efficiency<br />
when tested against a wide range of<br />
different sized supercoiled DNAs as well<br />
as DNA directly from a ligation reaction<br />
(see Table 1).<br />
Benefits<br />
• High transformation efficiency with<br />
clones of all sizes - including BAC clones<br />
(Table 1).<br />
• Lac ZΔM15 for blue/white screening of<br />
recombinants.<br />
• Restriction minus (mcrA, Δ(mrr-hsdRMSmcrBC))<br />
enables efficient cloning of<br />
methylated DNA.<br />
• Endonuclease minus (endA1) to ensure<br />
high yields of DNA.<br />
• Recombination minus (recA1) for greater<br />
stability of large cloned inserts.<br />
Genotype<br />
F - mcrA Δ(mrr-hsdRMS-mcrBC)<br />
φ80dlacZΔM15 ΔlacX74 recA1 endA1<br />
araD139 Δ(ara, leu)7697 galU galK λ- rpsL<br />
nupG<br />
TransforMax EC100 E. coli Transformation Efficiencies with Different DNAs<br />
DNA<br />
TransforMax EC100<br />
Electrocompetent E. coli<br />
• Transformation efficiency of >1 x 10 10<br />
cfu/µg of pUC19.<br />
TransforMax EC100<br />
Electrocompetent E. coli<br />
EC10005 5 X 100 µl<br />
EC10010 10 X 100 µl<br />
Includes pUC19 control DNA.<br />
TransforMax EC100<br />
Chemically Competent E. coli<br />
TransforMax EC100<br />
Electrocompetent E. coli<br />
pUC19 2.5 X 10 8 1.4 X 10 10<br />
8.1 kb Clone 1.3 X 10 7 Not tested<br />
13.1 kb Clone 4.3 X 10 6 1.3 X 10 9<br />
23.1 kb Clone 9.2 X 10 5 3.0 X 10 8<br />
<strong>14</strong>5 kb BAC Clone Not tested 7 X 10 7<br />
13.1 kb Clone directly<br />
from a ligation reaction<br />
2.2 X 10 5 2.1 X 10 7<br />
Table 1. Comparison of the<br />
transformation efficiencies<br />
of TransforMax EC100<br />
E. coli with a variety of<br />
DNAs. Transformations were<br />
performed using 50 µl of<br />
competent cells and either<br />
supercoiled DNAs of the<br />
indicated sizes or a 1 µl aliquot<br />
from a standard<br />
10 µl ligation reaction.<br />
Results shown are in cfu/µg<br />
of DNA and are the average<br />
transformation efficiencies<br />
obtained from several trials.<br />
TransforMax EC100 Chemically<br />
Competent E. coli<br />
• Transformation efficiency of >1 x 10 8<br />
cfu/µg of pUC19.<br />
• Supplied in convenient single-use 50 µl<br />
aliquots.<br />
TransforMax EC100 Chemically<br />
Competent E. coli<br />
CC02810 10 X 50 µl<br />
Includes pUC19 control DNA.<br />
Phage T1-resistant TransforMax EC100<br />
Competent cells are also available.<br />
Please visit www.EpiBio.com for details.<br />
Fast-Link DNA Ligation Kit<br />
DNA Ligations in 5 Minutes!<br />
5 min.<br />
15 min.<br />
30 min.<br />
1 hr.<br />
2 hr.<br />
<strong>EPICENTRE</strong>’s Fast-Link DNA Ligation Kit<br />
is formulated to provide fast and efficient<br />
DNA ligations in as little as 5 minutes at<br />
room temperature for both routine and<br />
high-throughput cloning.<br />
FIG 1. Time curve for cohesive-end ligation using Fast-<br />
Link Kit. Lambda Hind III markers were ligated using<br />
2 units of Fast-Link DNA Ligase (Lanes 3–7). Lane 1, kb<br />
ladder; Lane 2, no enzyme.<br />
Fast and Efficient DNA Ligations<br />
• Ligate cohesive-end DNA in 5 minutes<br />
at room temperature.<br />
• Ligate blunt-end DNA in 15 minutes at<br />
room temperature.<br />
• Ligation of PCR product with A-overhangs<br />
in 1 hour or less at 16°C.<br />
• Transform cells without desalting the<br />
ligation.<br />
1 2 3 4 5 6 7<br />
Fast-Link DNA Ligation Kit<br />
LK11025 25 Ligations<br />
LK0750H 50 Ligations<br />
LK6201H 100 Ligations<br />
Volume <strong>14</strong> • Number 2<br />
www.EpiBio.com<br />
23
The new TargetAmp Nano-g Biotin-aRNA Labeling Kit for the Illumina ® System produces<br />
Biotin-aRNA (also called cRNA) with high signal intensity for use with the Illumina ® Gene<br />
Expression System, such as the Illumina ® Expression BeadChips.<br />
TargetAmp Nano-g Biotin-aRNA Labeling Kit for the Illumina ® System—<br />
• Detects more genes using less input total RNA than another kit.<br />
• Easy, 6-hour linear RNA amplification and labeling process<br />
preserves the relative transcript abundance of the sample.<br />
• Optimized for producing Biotin-aRNA with high signal intensity<br />
from as little as 25 ng of input total RNA.<br />
See Page 9 for more details—Or visit www.EpiBio.com<br />
and enter code: TNAX1<br />
1656 7896 268<br />
Input RNA<br />
100 ng<br />
Input RNA<br />
400 ng<br />
• <strong>EPICENTRE</strong> • Another<br />
TargetAmp Nano-g Supplier’s Kit<br />
Biotin-aRNA Labeling Kit<br />
for the Ilumina ® System<br />
Transcripts detected using Biotin-aRNA (also<br />
called cRNA) produced by a TargetAmp <br />
Nano-g Biotin-aRNA Labeling Kit for the<br />
Illumina ® System. The kit detects more genes<br />
using less input total RNA than Biotin-aRNA<br />
produced by another kit.<br />
i2260807a<br />
NEW Products!<br />
QuickExtract DNA Extraction Solution<br />
TAQXpedite PCR Systems<br />
Now also<br />
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plants<br />
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See Pages 13 & <strong>14</strong> for more details—<br />
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and enter code: QEAX6<br />
See Pages 15 & 16 for more details—Or visit www.EpiBio.com<br />
and enter code: TGAX4<br />
P-FORUM<strong>14</strong>:2