State-of-the-art, ultra-high density BashingBeads are fracture resistant and chemically inert.

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1 INSTRUCTION MANUAL ZR Fecal DNA MiniPrep Catalog No. D6010 Highlights Rapid method for the isolation of inhibitor-free, PCR-quality DNA (up to 25 µg/prep) 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 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 Xpedition Sample Prep Technologies....6 List of Related Products For Research Use Only Ver

2 Page 1 Satisfaction of all Zymo Research products is guaranteed. If you should be dissatisfied with this product please call Product Contents ZR Fecal DNA MiniPrep (Kit Size) D6010 (50 preps.) Storage Temperature ZR BashingBead Lysis Tubes 50 Room Temp. Lysis Solution 40 ml Room Temp. Fecal DNA Binding Buffer* 100 ml Room Temp. DNA Pre-Wash Buffer** 15 ml Room Temp. Fecal DNA Wash Buffer 50 ml Room Temp. DNA Elution Buffer 10 ml Room Temp. Zymo-Spin IV Spin Filters (Orange Tops) 50 Room Temp. Zymo-Spin IV-HRC Spin Filters (Green Tops) 50 Room Temp. Zymo-Spin IIC Columns 50 Room Temp. Collection Tubes 200 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. * For optimal performance, add beta-mercaptoethanol to 0.5%(v/v) i.e., 500 µl per 100 ml. ** 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 150 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. A 260 /A 280 >1.8 DNA Size Limits Capable of recovering genomic DNA up to and above 40 kb. In most instances, mitochondrial DNA and viral DNA (if present) will also be recovered. DNA Recovery Typically, up to 25 µg total DNA is eluted into 100 µl (25 µl minimum) DNA Elution Buffer per sample. Equipment Microcentrifuge, vortex, cell disrupter/disrupter (recommended) Note - Trademarks of Zymo Research Corporation. This product is for research use only and should only be used by trained professionals. It is not intended for use in diagnostic procedures. 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. Disruptor Genie is a trademark of Scientific Industries, Inc. and FastPrep is a registered trademark of Qbiogene, Inc.

3 Page 2 Product Description The ZR Fecal DNA MiniPrep is designed for the simple, rapid isolation of inhibitor-free, 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 ( 150 mg each) are added directly to a ZR BashingBead Lysis Tube and rapidly and efficiently lysed by bead beating (e.g., Xpedition Sample Processor, page 6) 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, genotyping, etc. The DNA is also well suited for methylation profiling purposes using any of our EZ DNA Methylation family of products. A schematic of the ZR Fecal DNA MiniPrep procedure is shown below. Zymo Research offers the following for rapid, precise DNA methylation detection 1.) EZ DNA Methylation Kit (D5001, D5002, D5003) 2.) EZ DNA Methylation- Gold Kit (D5005, D5006, D5007, D5008) 3.) EZ DNA Methylation- Direct Kit (D5020, D5021, D5022, D5023)

4 Page 3 Zymo Research M Supplier Q Comparison of DNA yields from rat feces using the ZR Fecal DNA MiniPrep 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 MiniPrep. 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 For optimal performance, add beta-mercaptoethanol (user supplied) to the Fecal DNA Binding Buffer to a final dilution of 0.5%(v/v) i.e., 500 µl per 100 ml. 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 150 mg of fecal sample to a ZR BashingBead Lysis Tube. Add 750 µl Lysis Solution to the tube. 2. Secure in a bead beater fitted with a 2 ml tube holder assembly (e.g., Disruptor Genie ) and process at maximum speed for 5 minutes. Processing times may be as little as 40 seconds when using high-speed cell disrupters (e.g., the portable Xpedition Sample Processor, page 6, FastPrep -24, or similar). See manufacturer s literature for operating information. If the HRC matrix is dry, add µl water prior to prepping the filter. Cap tube tightly to prevent leakage. 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. Snap off the base of the Zymo-Spin IV Spin Filter prior to use. 5. Add 1,200 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 IIC Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute. The Zymo-Spin IIC Column has a maximum capacity of 800 µl. 7. Discard the flow through from the Collection Tube and repeat Step Add 200 µl DNA Pre-Wash Buffer to the Zymo-Spin IIC Column in a new Collection Tube and centrifuge at 10,000 x g for 1 minute. 9. Add 500 µl Fecal DNA Wash Buffer to the Zymo-Spin IIC Column and centrifuge at 10,000 x g for 1 minute. 10. Transfer the Zymo-Spin IIC Column to a clean 1.5 ml microcentrifuge tube and add 100 µl (25 µl minimum) 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.

6 Page 5 Ordering Information Product Description Catalog No. Kit Size ZR Fecal DNA MicroPrep D preps. ZR Fecal DNA MiniPrep D preps. ZR Fecal DNA MidiPrep D preps. ZR-96 Fecal DNA Kit D6011 2x96 preps. For Individual Sale Catalog No. Amount ZR BashingBead Lysis Tubes S Lysis Solution D ml 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) C Zymo-Spin IV-HRC Spin Filters (Green Tops) C Zymo-Spin IIC Columns C Collection Tubes Lysis Instruments C C C ,000 Description Cat. No. Amount Disruptor Genie, 120V w/ 2 ml tube holder assembly. Disruptor Genie, 240V w/ 2 ml tube holder assembly. S S unit 1 unit TurboMix Attachment, 2 ml Permanently mounts to most existing Vortex Genie mixers converting them to a Disruptor Genie. S unit The Disruptor Genie with 2 ml tube holder assembly from Scientific Industries, Inc. (Cat. No. S from Zymo Research Corp.)

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