DNA & RNA from Environmental Sources

Ultra-Pure DNA & RNAfrom Environmental SourcesEnvironmental samples (soil, sludge, sediment, feces, etc.) are rich sources of novel,uncharacterized microbes that are often unable to be cultured. This microbial diversity istypically difficult to study and hindered by the lack of rapid and robust purification schemesthat enable analysis. Similarly, samples refractory to conventional lysis procedures (such asplants, seeds, food, arthropods, yeast, filamentous fungi, unicellular and filamentous algae, andprotozoa) require efficient methods for the isolation of DNA and RNA.Canonical procedures for DNA and RNA isolation from these samples are often lengthy, messy,inconsistent and cost prohibitive.In addition, the purity of the DNA or RNA is not alwayssufficient for downstream applications in molecular biology. To address these issues, ZymoResearch has developed a range of technologies to easily and effectively extract DNA and RNAfrom these tough-to-lyse, difficult samples.The environmental DNA and RNA kits from Zymo Research feature chemically inert, high densityBashingBeads to thoroughly lyse samples and liberate nucleic acids for purification. Thisis coupled with unique Fast-Spin column and plate technologies to maximize yield and allowfor minimal volume elution in minutes. Polyphenolic compounds in environmental and plantsamples (i.e., humic/fulvic acids, tannins, etc.) can confound downstream analyses such as PCR. Our proprietary resins in select productsremove these compounds from the eluted DNA and RNA. This technology is also available as a stand-alone product for the cleanup ofimpure preparations as the OneStep PCR Inhibitor Removal Kit.At Zymo Research, we are committed to quality and guarantee that all of our products meet and exceed your expectations.USA: (888) 882-9682INTL: (949) 679-1190Fax: (949) 266-9452Find us on Facebook & follow uson Twitter for the latest productreleases, special offers, and more!Your questions, comments, and suggestions are always welcomeinfo@zymoresearch.comSign up to receive our newest publicationshttp://www.zymoresearch.com/publicationsPlease recycle this newsletter2

BashingBead Lysis forEnvironmental DNA PurificationThe BashingBead DNA purification kits from Zymo Research are for rapid recovery of inhibitor-free PCR-ready DNA from a broad range of tough-to-lyseorganisms and environmental samples. Kits have been specifically designed for the efficient recovery of inhibitor-free DNA from plants, seeds, tissues,insects, and microorganisms that inhabit soil, sludge, sediment, and fecal samples. Products are available in spin column Micro- (5 µg/prep), Mini- (25 µg/prep), Midi- (125 µg/prep) and 96-well (5 µg/well) formats – these formats are diagrammed below.For processing, samples are simply transferred into provided ZR BashingBead Lysis Tubes and efficiently lysed by bead beating in uniquely designedlysis buffers. Processing the samples can be performed using any bead mill, pulverizer, or vortex that can accommodate standard 2.0 ml, 50 ml tubes, or96-well blocks depending on the format of the kit. Following lysis, DNA is isolated using innovative Fast-Spin column and plate technologies, and in caseswhere plant, feces, or soil samples are processed, the DNA is subsequently filtered to remove humic/fulvic acids or polyphenols that can inhibit PCR. Theisolation of inhibitor-free DNA typically takes about 15 minutes.MicroPrep & MiniPrep FormatsMidiPrep Format96-Well Format((( )))$$$PCR-Ready Ultra-pure DNA$PCR-Ready, Ultra-pure DNAPulverize Samplewith ZR BashingBead Lysis TubeFilter Lysatewith Zymo-Spin IVContinue with the following step forZR Soil Microbe DNA, ZR Fecal DNA,and ZR Plant/Seed DNA Kits only.Bind, Wash, Elute DNAwith Zymo-Spin IC or IICPolyphenolic Removalwith Zymo-Spin IV-μHRC or IV-HRC((( )))$ $OR$$SpinWashElutePCR-Ready Ultra-pure DNAContinue with the following step forZR Soil Microbe DNA, ZR Fecal DNA,and ZR Plant/Seed DNA Kits only.$PCR-Ready, Ultra-pure DNAPulverize Samplein ZR BashingBead Lysis/Filtration TubeCentrifuge & Transfer Lysate toZymo-Spin V-E w/ Zymo-Midi Filter Polyphenolic Removalwith Zymo-Spin IV-HRC((( )))$$$PCR-Ready Ultra-pure DNAContinue with the following steps forZR Soil Microbe DNA, ZR Fecal DNA,and ZR Plant/Seed DNA Kits only.$PCR-Ready, Ultra-pure DNAPulverize Sampleswith ZR BashingBead Lysis RackDeep-well BlockBind, Wash, Elute DNAwith Silicon-A PlatePolyphenolic Removalwith Silicon-A -HRC Platewww.zymoresearch.com 3

= Spin ColumnApplication Guide: Environmental DNA PurificationZR Soil Microbe DNA Kits ZR Fungal/Bacterial DNA Kits ZR Fecal DNA Kits ZR Tissue & Insect DNA Kits ZR Plant/Seed DNA Kit= 96-WellMicroPrepMiniPrepMidiPrep 96-Well MicroPrepMiniPrepMidiPrep 96-Well MicroPrepMiniPrepMidiPrep 96-Well MicroPrepMiniPrepMidiPrep 96-Well MicroPrepMiniPrepMidiPrep 96-WellSpecificationsZR BashingBead Lysis P P P P P P P P P P P P P P P P P P P PFormatDNA Binding Capacity 5 µg 25 µg. 125 µg. 5 µg 5 µg. 25 µg. 125 µg. 5 µg 5 µg. 25 µg 125 µg. 5 µg 5 µg 25 µg 125 µg 5 µg 5 µg 25 µg 125 µg 5 µgElution Volume ≥ 10µl ≥ 25µl ≥ 150µl ≥ 25µl ≥ 10µl ≥ 25µl ≥ 150µl ≥ 25µl ≥ 10µl ≥ 25µl ≥150 µl ≥ 25µl ≥ 10µl ≥ 25µl ≥ 150µl ≥ 25µl ≥ 10µl ≥ 25µl ≥ 150µl ≥ 25 µlRemoval of PCR Inhibitors P P P P P P P P P P P P P P P P P P P PRemoval of Humic, Fulvic,Polyphenolic SubstancesP P P P P P P P P P P PProcessing Time 15 min. 15 min. 25 min. 50 min. 10 min. 10 min. 20 min. 40 min. 15 min. 15 min. 25 min. 50 min. 10 min. 10 min. 20 min. 40 min. 15 min. 15 min. 25 min. 50 min.ApplicationEnvironmental SourcesSoil P P P PSediment P P P PSludge P P P PFeces P P P PMicroorganismsBacteria P P P P P P P P P P P PGram (+) P P P P P P P P P P P PGram (-) P P P P P P P P P P P PFungi P P P P P P P P P P P PUnicellular (Yeast) P P P P P P P P P P P PFilamentous P P P P P P P P P P P PAlgae P P P P P P P P P P P PUnicellular P P P P P P P P P P P PFilamentous P P P P P P P P P P P PProtists P P P P P P P P P P P PTough-to-Lyse Tissues, Organisms, Insects, & PlantsSoft Tissues Some Some Some Some Some Some Some Some Some Some Some Some P P P PSolid Tissues (Food) P P P PTough-to-Lyse Tissues P P P PTough-to-Lyse Organisms P P P PInsects/Arthropods P P P PPlant Material P P P PSeeds P P P PFruit P P P P

ZR Soil Microbe DNA KitsHighlights Simple, efficient isolation of humic-free DNA from microbes in soil, sludge, sediment, and sand in minutes.Ideal for tough-to-lyse bacteria, fungi, algae, and protozoa. Fast-Spin column and unique filtration technologies effectively remove PCR inhibitors from the DNAproduct.SamplesAB1 kb 1 2 3 4 5APPLICATIONPPPPPPPPPSoilSedimentSludgeGram (+) BacteriaGram (-) BacteriaYeastFilamentous FungiUnicellular AlgaeFilamentous AlgaeSample 1Class: Sand

ZR Fecal DNA KitsHighlights Rapid methods for the isolation of inhibitor-free, PCR-quality DNA from fecal samples in minutes includingthose from humans, birds, rats, mice, cattle, etc. Fast-Spin column and unique filtration technologies effectively remove PCR inhibitors from the DNAproduct.APPLICATIONP FecesP Bacteria Gram (+)P Bacteria Gram (-)P YeastP Filamentous FungiP Unicellular AlgaeP Filamentous AlgaeP ProtistCat # Desc. PrepsD6012 Micro 50D6010 Mini 50D6110 Midi 25D6011 96-Well 2 x 96ZymoOther SuppliersResearch M QComparison of DNA yields fromrat feces using the ZR Fecal DNAMiniPrep and kits from suppliers Mand Q. Equivalent amounts of feces wereprocessed using each kit and then equalvolumes of eluted DNA were analyzed in a0.8% (w/v) agarose/ethidium bromide gel.Samples were processed in triplicate.Comparative Overview►Fecal Sample OnlyFecal Sample + E. coli (pUC19)M 1 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 1 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6The results of PCR of DNA isolated from human feces with and without theaddition of E. coli containing pUC19 plasmid DNA (indicated at the top of theimage) using primers specific for the pUC19 sequence. In each case, ampliconswere analyzed in a 1.5% (w/v) agarose/ethidium bromide gel using a UV imager.Numbers above each lane of the gel images are the volumetric equivalent (in µl) ofeluted DNA used for PCR. Arrows mark the relative migration of amplicons in the gels,and M is a 100 bp DNA ladder (NEB).ZR Fecal DNA MiniPrep Supplier M Supplier QProcessing Time 10-15 minutes 40-50 minutes 40-50 minutesDNA Yield High Fair LowDNA Integrity Good Fair PoorFidelity of PCR High High FairUse of Proteinases No No YesUse of Organic Denaturants No No NoZR Tissue & Insect DNA KitsHighlight Simple and efficient isolation of DNA from insects, including mosquitoes, bees, lice, ticks, and D.melanogaster. Also compatible with tough-to-lyse tissues from other organisms.APPLICATIONP Soft & Solid Tissues (Food)P Tough-to-Lyse Tissues1 kbD. melanogaster n = 10D. melanogaster n = 20D. melanogaster larvae n = 10D. melanogaster larvae n = 20Darkling Beetle larvaCricket1 kbMouse KidneyMouse LiverMouse TailsnipP Tough-to-Lyse OrganismsP Insects/ArthropodsCat # Desc. PrepsD6015 Micro 50D6016 Mini 50D6115 Midi 25D6017 96-Well 2 x 96DNA yields from various insect and mouse samplesusing the ZR Insect & Tissue DNA MiniPrep . Variousamounts of sample were processed with equal volumesof eluted DNA analyzed in a 0.8% (w/v) agarose/ethidiumbromide gel. The 1 kb DNA size marker is from ZymoResearch.6www.zymoresearch.com

ZR Soil/Fecal RNA KitsHighlights Simple and efficient method for the isolation of inhibitor-free RNA from soil and fecal samples. Ultra-high density BashingBeads can be used with any bead mill, disrupter, or vortex.MRT-PCR+HRC -HRCControlAPPLICATIONPPPPSoilSedimentSludgeFecesCat # Desc. PrepsR2040 Micro 50PCR amplification of a eukaryotic transcriptpost-RT: Total RNA isolated from sludge with orwithout inclusion of the Zymo-Spin IV-HRC spinfilter during the ZR Soil/Fecal RNA MicroPrep protocol. M is a ZR 1 kb DNA Marker (ZymoResearch).ZR Fungal/Bacterial RNA KitsHighlights Quick (15 minute) isolation of total RNA from tough-to-lyse bacteria, yeast, and fungi. Omits the use of organic denaturants and proteases.APPLICATIONP Gram ( + ) BacteriaP Gram ( - ) BacteriaP YeastP Filamentous Fungi9.0 -6.0 -3.0 -2.0 -1.0 -RNALadderZymoResearch Supplier Q DNALadder- 10.0- 3.0- 1.0- 0.5P Unicellular AlgaeP Filamentous AlgaeP ProtistsP Soft Tissues (limited)P FoodCat # Desc. PrepsR2010 Micro 50R2014 Mini 500.5 -kbDNase I * DNase ITotal RNA was isolated from equal amounts of pelleted E. coli cells containingplasmid DNA (pGEM ® ) using the ZR Fungal/Bacterial RNA MicroPrep or thekit from Supplier Q. The samples were resolved in a 2% (w/v) agarose gel.RNA Millenium Markers (Ambion) and ZR 1 kb DNA Marker (Zymo Research)were used.* = genomic (> 10 kb) and plasmid (> 3 kb) DNA contamination; DNase I = samplestreated with DNase I.kbwww.zymoresearch.com 9

ZR Tissue Insect RNA KitsHighlights Quick (15 minute) isolation of RNA from insects and tough-to-lyse tissues. Omits the use of organic denaturants and proteases.APPLICATIONP Soft TissuesP Solid TissuesP Tough-to-Lyse TissuesP Tough-to-Lyse OrganismsP Insects/ArthropodsP FoodCat # Desc. PrepsR2030 Micro 509.0 -6.0 -5.0 -4.0 -3.0 -2.5 -2.0 -1.5 -1.0 -kbM 1 2Analysis of ZR Tissue & Insect RNA MicroPrep .Isolation of total RNA from n=2 Drosophila sp.individuals was performed in duplicate (lanes 1 and2). Samples were processed (2 x 30 sec at 6 m/s)using a FastPrep ® -24 Instrument (MP Biomedicals)and resolved alongside (lane M) RNA Millenium Markers (Ambion) in a 1% (w/v) non-denaturingagarose gel.ZR Plant RNA KitsHighlights Quick (15 minute) isolation of inhibitor-free total RNA from a variety of plant tissues. Fast-spin column technology coupled with filtration removes polyphenolic RT inhibitors from the RNAproductSample 1 Sample 2APPLICATIONP Plant MaterialP SeedsP FruitCat # Desc. PrepsR2024 Mini 50Isolation of total RNA from 10 mg of a fresh leafmaterial (Nicotiana sp.) using the ZR Plant RNAMiniPrep . Leaves were minced then processed usinga FastPrep ® -24 instrument (MP Biomedicals). Samples1 and 2 were loaded in 2x and 1x volume aliquots,respectively, and resolved in a 1% (w/v) nondenaturingagarose gel. RNA Millenium Markers (Ambion) wereused as size standards.10www.zymoresearch.com

OneStep PCR Inhibitor Removal KitsHighlights Removes PCR inhibitors such as polyphenolics, humic/fulvic acids, tannins, melanin, etc. from nucleicacid solutions to yield high quality DNA or RNA. Fast, one-step procedure for cleaning impure samples prior to PCR, sequencing, reverse transcription(RT), etc.APPLICATIONPPPolyphenolic PCR InhibitorRemoval from DNAPolyphenolic RT InhibitorRemoval from RNACat # Desc. PrepsD6030SpinColumn50D6035 96-Well 2 x 96OneStep PCR Inhibitor Removal Kit ProcedureSpin Column Format96-Well FormatAdd Sample300 bp -200 bp -100 bp -LadderControlsDNA Sample Containing Humic AcidNon-TreatedTreated ProductDNA is efficiently amplified by PCR following humic acid removalwith the OneStep PCR Inhibitor Removal Kit. The figure showsamplification of a 200 bp product from DNA containing humic acid thatwas treated with the kit. Alternatively, PCR amplification was completelyinhibited in the case of the non-treated sample. In each case, equalamounts of DNA were used for each PCR and equivalent amounts of thereaction were then analyzed in a 2.0% (w/v) agarose/TAE/EtBr gel. Theladder is a 100 bp DNA marker (Zymo Research). Hot start PCR wasperformed using ZymoTaq PreMix (Zymo Research).$M+HRCRT-PCR-HRCControlInhibitor-Free DNA (or RNA)PCR amplification of an eukaryotic transcript (post-RT): Total RNAisolated from sludge with or without inclusion of the Zymo-Spin IV-HRCSpin Filter. M is a 1 kb DNA Marker (Zymo Research).ZR BashingBead Lysis Tubes & RacksZR BashingBead Lysis Tubes (0.5 mm or 2 mm) Each impact resistant 2.0 ml tube contains 0.7 ml (dry volume) of ZR BashingBead lysis matrix, availablein 0.5 or 2 mm. These state-of-the-art, ultra-high density beads are fracture resistant and chemically inert and ideal fordisrupting tough-to-lyse bacteria, yeast, fungi, and algae.Cat # Desc. Qty.S6002-50 Tube(0.5 mm)S6003-50 Tube(2.0 mm)S6002-96-1S6002-96-2Rack(0.5 mm)Rack(2.0 mm)505011ZR-96 BashingBead Lysis Rack (0.5 mm or 2 mm) Each impact resistant 1.1 ml tube contains 0.5 ml dry volume of ZR BashingBead lysis matrix, availablein 0.5 or 2 mm. Tubes are in a 96-well rack with caps and a cover for high throughput processing. Perfect for microbes and fungi in soil, feces, sludge, etc.www.zymoresearch.com 11

Environmental DNAD6003 ZR Soil Microbe DNA MicroPrep 50 preps.D6001 ZR Soil Microbe DNA MiniPrep 50 preps.D6101 ZR Soil Microbe DNA MidiPrep 25 preps.D6002 ZR-96 Soil Microbe DNA Kit 2 x 96 preps.D6007 ZR Fungal/Bacterial DNA MicroPrep 50 preps.D6005 ZR Fungal/Bacterial DNA MiniPrep 50 preps.D6105 ZR Fungal/Bacterial DNA MidiPrep 25 preps.D6006 ZR-96 Fungal/Bacterial DNA Kit 2 x 96 preps.D6012 ZR Fecal DNA MicroPrep 50 preps.D6010 ZR Fecal DNA MiniPrep 50 preps.D6110 ZR Fecal DNA MidiPrep 25 preps.D6011 ZR-96 Fecal DNA Kit 2 x 96 preps.D6015 ZR Tissue & Insect DNA MicroPrep 50 preps.D6016 ZR Tissue & Insect DNA MiniPrep 50 preps.D6115 ZR Tissue & Insect DNA MidiPrep 25 preps.D6017 ZR-96 Tissue & Insect DNA Kit 2 x 96 preps.D6022 ZR Plant/Seed DNA MicroPrep 50 preps.D6020 ZR Plant/Seed DNA MiniPrep 50 preps.D6120 ZR Plant/Seed DNA MidiPrep 25 preps.D6021 ZR-96 Plant/Seed DNA Kit 2 x 96 preps.Environmental RNAR2040 ZR Soil/Fecal RNA MicroPrep 50 preps.R2010 ZR Fungal/Bacterial RNA MicroPrep 50 preps.R2014 ZR Fungal/Bacterial RNA MiniPrep 50 preps.R2030 ZR Tissue & Insect RNA MicroPrep 50 preps.R2024 ZR Plant RNA MiniPrep 50 preps.Cell Disrupters & AccessoriesDisruptor Genie ® S6001-2-230 European Plug 1 unitS6001-2-120 120V 1 unitBullet Blender S6007-2 BBX24B Bullet Blender Blue (24 x 1.5 - 2.0 ml tubes) with cooling fan 1 unitS6007-1 BBX24 Bullet Blender (24 x 1.5 - 2.0 ml tubes) 1 unitS6007-3 BBX50B Bullet Blender Blue 50 (9 x 50 ml tubes) with cooling fan 1 unitFastPrep ® -24 S6005 FastPrep ® -24 1 unitFastPrep ® -24 AcessoriesS6005-1 HiPrep Attachment (48 x 2 ml tubes) 1 unitS6005-2 CoolPrep Attachment (24 x 2 ml tubes) 1 unitS6005-3 TeenPrep Attachment (12 x 15 ml tubes) 1 unitS6005-4 BigPrep Attachment (2 x 50 ml tubes) 1 unitS6005-5 FastPrep ® European AC Cord 1 unit2010 Geno/Grinder ® S6006 2010 Geno/Grinder ® 1 unit2010 Geno/Grinder ®AccessoriesS6006-1 2 ml Tube Holder/Cryo Block Assembly (48 x 2.0 ml tubes/block) 2 blocksS6006-2 15 ml Tube Holder/Cryo Block Assembly (15 x 15 ml tubes/block) 2 blocksS6006-3 50 ml Tube Holder/Cryo Block Assembly (6 x 50 ml tubes/block) 2 blocksS6006-10 Large Capacity Clamp Assembly 1 unitalso availableD6030 OneStep PCR Inhibitor Removal Kit 50 preps.D6035OneStep-96 PCR Inhibitor RemovalKit2 x 96 preps.S6002-50ZR BashingBead Lysis Tubes(0.5 mm)50 tubesS6003-50S6002-96-1ZR BashingBead Lysis Tubes(2.0 mm)ZR-96 BashingBead Lysis Rack (0.5mm)50 tubes1 rackFor a complete product listing,please visit www.zymoresearch.comS6002-96-2ZR-96 BashingBead Lysis Rack (2.0mm)1 rackFind us on Facebook & follow us on Twitter for the latestproduct releases, special offers, and more!