INSTRUCTION MANUAL. ZR Fecal DNA Kit Catalog No. D6010. Highlights. Contents

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1 INSTRUCTION MANUAL ZR Fecal DNA Kit Catalog No. D6010 Highlights Rapid method for the isolation of inhibitor-free, PCR-quality DNA from fecal samples in as little as 15 minutes including humans, birds, rats, mice, cattle, etc. State-of-the-art, ultra-high density BashingBeads are fracture resistant and chemically inert. Can be used with any bead mill, disrupter, or vortex that can accommodate standard 2.0 ml tubes. Omits the use of organic denaturants as well as proteinases. Contents Product Contents & Specifications..1 Product Description...2, 3 Protocol 4 Ordering Information..5 List of DNA-Related Products For Research Use Only Ver

2 Page 1 Product Contents ZR Fecal DNA Kit (Kit Size) D6010 ( preps.) Storage Temperature ZR BashingBead Lysis Tubes tubes Room Temp. Fecal DNA Binding Buffer 100 ml Room Temp. Note: Satisfaction of all Zymo Research products is guaranteed. If you should be dissatisfied with this product please call DNA Pre-Wash Buffer* 15 ml Room Temp. Fecal DNA Wash Buffer ml Room Temp. DNA Elution Buffer 10 ml Room Temp. Zymo-Spin IV Spin Filters (Orange Tops) filters Room Temp. Zymo-Spin IV-HRC Spin Filters (Green Tops) filters Room Temp. Zymo-Spin II Columns columns Room Temp. Collection Tubes tubes Room Temp. Instruction Manual 1 - Note - Integrity of kit components is guaranteed for up to one year from date of purchase. Reagents are routinely tested on a lot-to-lot basis to ensure they provide maximal performance and reliability. * A precipitate may have formed in the DNA Pre-Wash Buffer during shipping. To completely resuspend the buffer, incubate the bottle at ºC for 30 minutes and mix by inversion. DO NOT MICROWAVE. Specifications Format Bead Beating, Spin Column. Sample Sources Bacterial, protist, as well as host DNA can be effectively isolated from a 1 mg sample of mammalian feces. DNA Purity High quality, inhibitor-free DNA is eluted with DNA Elution Buffer suitable for the amplification of bacterial, protist, and/or mammalian templates. Absorbance 260 nm/280 nm > 1.8 DNA Size Limits > 1 kb. DNA Recovery Typically, up to 25 µg total DNA is eluted into 100 µl (25 µl minimum) DNA Elution Buffer per sample. For DNA 75 bp to 10 kb, the recovery is 70-90%. For DNA 11 kb to 23 kb the recovery is -70%. Equipment Microcentrifuge, vortex, cell disrupter/disrupter (optional) Note - Trademarks of Zymo Research Corporation. This product is for research use only and should only be used by trained professionals. Some reagents included with this kit are irritants. Wear protective gloves and eye protection. Follow the safety guidelines and rules enacted by your research institution or facility.

3 Page 2 Product Description The ZR Fecal DNA Kit is designed for the simple, rapid isolation of inhibitorfree, PCR-quality DNA from a variety of sample sources including humans, birds, rats, mice, cattle, etc. The procedure is easy and can be completed in as little as 15 minutes: fecal samples ( 1 mg each) are added directly to a ZR BashingBead Lysis Tube and rapidly and efficiently lysed without the use of organic denaturants or proteinases. The DNA is then isolated and purified using our Fast-Spin column technology and is ideal for downstream molecular-based applications including PCR, arrays, etc. The DNA is also well suited for methylation profiling purposes using any of our EZ DNA Methylation Kits. A schematic of the ZR Fecal Kit procedure is shown below.

4 Page 3 Zymo Research M Supplier Q Comparison of DNA yields from rat feces using the ZR Fecal DNA Kit and kits from suppliers M and Q. Equivalent amounts of feces were processed using each kit and then equal volumes of eluted DNA were analyzed in a 0.8% (w/v) agarose/ethidium bromide gel. Samples were processed in triplicate. A M M B C Fecal Sample Only Fecal Sample + E. coli(puc19) M PCR of DNAs from rat and human fecal samples isolated with the ZR Fecal DNA Kit. Panels A and B show the results of PCR with DNA isolated from rat and human fecal samples, respectively, using primers specific for prokaryotic 16S rrna. Panel C shows the results of PCR of DNA isolated from human feces with and without the addition of E. coli containing puc19 plasmid DNA (indicated at the top of the image) using primers specific for the puc19 sequence. In each case, amplicons were 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) of eluted DNA (100 µl) used for PCR. Arrows mark the relative migration of amplicons in the gels, and M is a 100 bp DNA ladder (NEB). For Technical Assistance, please contact those at Zymo Research s Technical Department at or to tech@zymoresearch.com.

5 Page 4 Protocol Before Starting: Zymo-Spin IV-HRC Spin Filters (green tops) need to be prepared prior to use by: 1) snapping off the base, 2), inserting into a Collection Tube, and 3), spinning in a microcentrifuge at exactly 8,000 x g for 3 minutes. 1. Add up to 1 mg of fecal sample to a ZR BashingBead Lysis Tube. 2. Secure in a bead beater fitted with a 2.0 ml tube holder assembly (e.g., Scientific Industries Disruptor Genie, Cat. No. S from Zymo Research Corp.) and process at maximum speed for 5 minutes. Processing times may be as little as 1 minute when using high-speed cell disrupters (e.g., FastPrep and Precellys ). See manufacturers literature for operating information. Note: Cap tube tightly to prevent leakage. Note: Alternatively, a standard bench top vortex can be used although the overall yield of DNA may be lower. 3. Centrifuge the ZR BashingBead Lysis Tube in a microcentrifuge at 10,000 x g for 1 minute. 4. Transfer up to 400 µl supernatant to a Zymo-Spin IV Spin Filter (orange top) in a Collection Tube and centrifuge at 7,000 rpm (~7,000 x g) for 1 minute. Note: Snap off the base of the Zymo-Spin IV Spin Filter prior to use. 5. Add 1, µl of Fecal DNA Binding Buffer to the filtrate in the Collection Tube from Step Transfer 800 µl of the mixture from Step 5 to a Zymo-Spin II Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute. Note: The Zymo-Spin II Column has a maximum capacity of 800 µl. 7. Discard the flow through from the Collection Tube and repeat Step Add µl DNA Pre-Wash Buffer to the Zymo-Spin II Column in a new Collection Tube and centrifuge at 10,000 x g for 1 minute. 9. Add 0 µl Fecal DNA Wash Buffer to the Zymo-Spin II Column and centrifuge at 10,000 x g for 1 minute. 10. Transfer the Zymo-Spin II Column to a clean 1.5 ml microcentrifuge tube and add 100 µl DNA Elution Buffer directly to the column matrix. Centrifuge at 10,000 x g for 30 seconds to elute the DNA. 11. Transfer the eluted DNA from Step 10 to a prepared Zymo-Spin IV-HRC Spin Filter (green top) (see above) in a clean 1.5 ml microcentrifuge tube and centrifuge at exactly 8,000 x g for 1 minute. The filtered DNA is now suitable for PCR and other downstream applications. Disruptor Genie is a trademark of Scientific Industries, Inc. FastPrep is a registered trademark of Qbiogene, Inc. Precellys is a registered trademark of Bertin Technologies

6 Page 5 Ordering Information Product Description Catalog No. Kit Size ZR Fecal DNA Kit D6010 preps. For Individual Sale Catalog No. Amount BashingBead Lysis Tubes (dry beads only) S6002- tubes Fecal DNA Binding Buffer D ml DNA Pre-Wash Buffer D ml Fecal DNA Wash Buffer D ml DNA Elution Buffer D ml Zymo-Spin IV Spin Filters (Orange Tops) C1007- filters Zymo-Spin IV-HRC Spin Filters (Green Tops) C1010- filters Zymo-Spin II Columns Collection Tubes Disruptor Genie, 120V w/ 2.0 ml Tube Holder Assembly TurboMix Attachment, 2.0 ml (Permanently mounts to most existing Vortex-Genie 2 and Vortex-Genie 2T mixers converting them to a Disruptor Genie. C1008- C C1001- C C S unit S unit columns 2 columns tubes 0 tubes 1,000 tubes The Disruptor Genie with 2.0 ml Tube Holder Assembly from Scientific Industries, Inc. (Cat. No. S from Zymo Research Corp.)

7 Page 6 Popular DNA Purification & Analysis Products from Zymo Product Concentrator -5 Concentrator -25 Concentrator -100 Concentrator -0 ZR-96 DNA Clean & Concentrator -5 Zymoclean Gel DNA Recovery Kit ZR-96 Zymoclean Gel DNA Recovery Kit Pinpoint Slide DNA Isolation System Zyppy Plasmid Miniprep Kit Zyppy Plasmid Midiprep Kit Zyppy Plasmid Maxiprep Kit ZR Genomic DNA I Kit ZR Genomic DNA II Kit ZR-96 Genomic DNA Kit Description Clean & concentrate DNA from any reaction or crude preparation in 2 minutes. A 6 µl minimum elution volume allows for highly concentrated DNA. Designed for samples containing up to 5 µg of DNA. Clean & concentrate DNA in minutes. 25 µl minimum elution volume allows for highly concentrated DNA. Designed for purifying up to 25 µg of DNA. Clean & concentrate DNA in minutes. 100 µl minimum elution volume allows for highly concentrated DNA. Designed for purifying up to 100 µg of DNA. Clean & concentrate DNA in minutes. 1 ml minimum elution volume allows for highly concentrated DNA. Designed for samples containing up to 0 µg of DNA. Quick (15 minute), high-output recovery of pure DNA from PCR, endonuclease digestions, plasmid preparations, etc µl minimum elution volume allows for highly concentrated DNA. Designed for samples containing up to 5 µg of DNA. Kit Size (Preps.) Purify DNA from high and low-melting agarose gels in minutes High-throughput DNA purification from high and low-melting agarose gels. Recover genomic DNA from paraffin-embedded or fresh tissue sections for PCR. Ideal for isolating DNA from clinical tissue samples. High-purity plasmid DNA purification in minutes: (alkaline lysis/spin column format for low 25 µl elution volume). High-purity plasmid DNA purification in minutes: (alkaline lysis/spin column format and 1 µl minimum elution volume). High-purity plasmid DNA purification in minutes: (alkaline lysis/spin column format and 2 ml minimum elution volume). Genomic DNA isolation from whole blood, tissue culture cells, solid tissue and liquid samples. (Silica bead format is scalable to fit your requirements). Genomic DNA purification from whole blood, tissue culture cells, solid tissue and liquid samples. No requirement for beads or phenol chloroform. High-output genomic DNA purification from whole blood, tissue culture cells, solid tissue and liquid samples. No requirement for beads or phenol chloroform. Catalog No. (Column Format) D4003 (uncapped) D4004 (uncapped) D4013 (capped) D4014 (capped) D4005 D4006 D4029 D4030 D4031 D4032 D4023 D4024 D4001 D4002 D4021 D4022 D D4019 D4020 D4025 D4026 D4019 D4020 D3004 D3005 D3006 (uncapped) D3007 (uncapped) D3024 (capped) D3025 (capped) D3010 D3011 ZR Soil Microbe DNA Kit Simple, rapid isolation of humic-free, PCR-quality genomic DNA from soil microbes. D6001 ZR Fungal/Bacterial Simple, rapid isolation of PCR-quality genomic DNA from fungi. D6005 DNA Kit ZR Fecal DNA Kit Simple, rapid isolation of PCR-quality genomic DNA from feces. D6010 ZR Viral DNA Kit ZR-96 Viral DNA Kit EZ DNA Methylation Kit Isolation of viral DNA from cell-free body fluids or sample mixtures containing cells at concentrations less than 10 5 cells per ml. High-output (96-well) isolation of viral DNA from cell-free body fluids or sample mixtures containing cells at concentrations less than 10 5 cells per ml. Streamlined kit for the conversion of unmethylated cytosines in DNA to uracil via the chemical-denaturation of DNA using our specially designed CT Conversion Reagent. DNA is then desulphonated and subsequently cleaned using Fast-Spin column technology. Ultra-pure recovered DNA can be used for PCR and bisulfite sequencing applications. D3015 D3016 D3017 D3018 D01 D02 D03 EZ DNA Methylation-Gold Kit Streamlined kit for the conversion of unmethylated cytosines in DNA to uracil via heat-denaturation of DNA using our specially designed CT Conversion Reagent. DNA is then desulphonated and subsequently cleaned using Fast-Spin column technology. Ultra-pure recovered DNA can be used for PCR and bisulfite sequencing applications. 3 hour processing time! D05 D06 D07 *Bulk quantities are available upon request. Please contact: busdev@zymoresearch.com or call for assistance.