INSTRUCTION MANUAL Xpedition Soil/Fecal DNA MiniPrep Catalog No. D6202 Highlights Take the Lab to the Field with state of the art Xpedition Sample Prep and DNA Preservation Technologies. For efficient BashingBead lysis and isolation of humic-free, PCR-quality DNA from tough-to-lyse bacteria, fungi, algae, and protozoa in soil, sand, sludge, sediment, and feces. Unique lysis/stabilization solution keeps DNA intact in lysates for storage and transport following remote sample processing with the Xpedition Sample Processor (XSP). Contents Product Contents & Specifications... 1 Product Description... 2,3 Protocol... 4 Ordering Information... 5 Xpedition Sample Prep Technologies... 6 List of Related Products... 6 For Research Use Only Ver. 1.0.0
Page 1 Satisfaction of all Zymo Research products is guaranteed. If you should be dissatisfied with this product please call 1-888- 882-9682. Product Contents Xpedition Soil/Fecal DNA MiniPrep (Kit Size) D6202 (50 preps.) Storage Temperature ZR BashingBead Lysis Tubes 50 Room Temp. Xpedition Lysis/Stabilization Solution 40 ml Room Temp. Soil/Fecal DNA Binding Buffer* 100 ml Room Temp. DNA Pre-Wash Buffer** 15 ml Room Temp. Soil/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 IIC Columns 50 Room Temp. Zymo-Spin IV-HRC Spin Filters (Green Tops) 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 30-37 ºC for 30 minutes and mix by inversion. DO NOT MICROWAVE. Specifications Format Bead Beating, Spin Column Purification Sample Sources Fungi, protozoa, algae, etc. in up to 0.25 g of soil, feces, dirt, sediment, and sludge. DNA Purity High quality, polyphenolic-free DNA is eluted with DNA Elution Buffer making it perfect for PCR. A 260 /A 280 >1.8 DNA Preservation/Stabilization Generally, DNA is preserved and stable in soil/fecal lysates at ambient temperatures for up to a month in Xpedition Lysis/Stabiliztion Solution. For prolonged preservation, store at -20 to -70 o C. 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, Portable Disruptor/Bead Beater (Optional) 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. Some Xpedition technologies are patent pending.
Page 2 Product Description Degradation and contamination of biological samples have been obstacles to scientific study, and may be particularly problematic in highly sensitive molecularanalysis techniques (e.g., PCR of low copy DNA). Use of cryogenic freezing methods for environmental/forensic sample preservation may often be too impractical to be employed. The Xpedition Soil/Fecal DNA MiniPrep, when used with a portable bead beating device [i.e., Xpedition Sample Processor (XSP), pg. 6], allows the researcher/investigator to Take the Lab to the Field. The Xpedition Soil/Fecal DNA MiniPrep can be used for the isolation of inhibitor-free DNA from tough-to-lyse bacteria, fungi, protozoa, algae, etc., that inhabit a range of samples including feces as well as clay, sandy, salty, silty, peaty, chalky, and loamy soils. Samples from remote locations can be processed at the site of collection using the provided ZR BashingBead Lysis Tubes and a XSP (available separately, Cat. No. S6020, pg. 6). Sample processing occurs in a unique lysis/stabilization solution that effectively preserves DNA in soil/fecal lysates for subsequent storage and transport. Later, when convenient with the researcher, the kit s Fast-Spin column technologies are used for the isolation of humic acid/polyphenol-free DNA from the lysates. This product can also be used for isolation of DNA from cultured bacteria, fungi, and yeast. Eluted DNA is ideal for PCR for various genotypic analysis including metagenomics, forensics, barcoding, phylogenetics, etc. An overview of the procedure is shown below.
Page 3 For Technical Assistance, please contact those at Zymo Research s Technical Department at 1-888-882-9682 or E-mail to tech@zymoresearch.com. Sample Preservation Stabilization of DNA in Sample Lysates for Storage/Transport Fig. 1. DNA in lysates is effectively stabilized for non-refrigerated storage/transport using the Xpedition Lysis/Stabilization Solution. For both soil and liver samples: lysates were generated using the Xpedition Sample Processor and stored at room temperature over the course of two weeks prior to DNA isolation using the appropriate Xpedition kit (pg. 6). Equal volumes of eluate were subsequently visualized in 0.8% (w/v) agarose/ethidium bromide gels (shown above). The size marker (M) is a 1 kb ladder (Zymo Research). Also, PCR amplicons generated from final time points are shown in gel images to the right as indicated. High Yield, Inhibitor-Free DNA Isolation M Fig. 2. The Xpedition Sample Processor effectively lyses tough-to-lyse biological materials for subsequent high yield, inhibitor-free DNA isolation. Two (2) weeks following sample processing with the XSP, DNA was isolated from plant, soil, mouse, and insect tissue lysates using specific Zymo Research environmental DNA kits (pg. 6) and then separated in a 0.8% (w/v) TAE/agarose/EtBr gel. DNA from source material is graphically represented above the gel image and M is a 1 kb molecular weight DNA marker (Zymo Research). Inhibitor-free DNA is an ideal template for direct PCR as evidenced in Fig. 1 (above).
Page 4 Protocol For optimal performance, add beta-mercaptoethanol (user supplied) to the Soil/Fecal DNA Binding Buffer to a final dilution of 0.5%(v/v) i.e., 500 µl per 100 ml. (Soil/Fecal samples only) 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 0.25 grams of soil or fecal material sample to a ZR BashingBead Lysis Tube. Add 750 µl Xpedition Lysis/Stabilization Solution to the tube. Notes: If the HRC matrix is dry, add 400-600 µl water prior to prepping the filter. Cap tube tightly to prevent leakage. Alternatively, add 50-100 mg (wet weight) fungal and/or bacterial cells that have been resuspended in up to 200 µl of water or isotonic buffer (e.g., PBS) to a ZR BashingBead Lysis Tube. 2. Secure in a bead beater fitted with a 2 ml tube holder assembly (e.g., Xpedition Sample Processor for processing at the site of sample collection) and process for a minimum of 30 seconds. Store/transport samples. Proceed to Step 3 when convenient. 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 x g (~7,000 rpm) for 1 minute. Snap off the base of the Zymo-Spin IV Spin Filter prior to use. 5. Add 1,200 µl of Soil/Fecal DNA Binding Buffer to the filtrate in the Collection Tube from Step 4. 6. 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. 7. Discard the flow through from the Collection Tube and repeat Step 6. The Zymo-Spin IIC Column has a maximum capacity of 800 µl. 8. 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 Soil/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. If fungi or bacterial cultures were sampled, the DNA is now suitable for PCR as well as other downstream applications. In some cases a browncolored pellet may form at the bottom of the tube after centrifugation. Avoid this pellet when collecting the eluted 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.
Page 5 Ordering Information There is a Xpedition DNA MiniPrep Kit for Catalog No. Kit Size Soil & Feces D6202 50 preps. Fungus & Bacteria D6206 50 preps. Tissue, Insects & Food D6216 50 preps. Plants, Seeds & Fruit D6221 50 preps. For Individual Sale Catalog No. Amount ZR BashingBead Lysis Tubes S6002-50 50 Xpedition Lysis/Stabilization Solution D6202-1-40 40 ml Soil/Fecal DNA Binding Buffer D6202-2-100 100 ml DNA Pre-Wash Buffer D3004-5-15 15 ml Soil/Fecal DNA Wash Buffer D6202-3-50 50 ml DNA Elution Buffer D3004-4-10 10 ml Zymo-Spin IV Spin Filters (Orange Tops) C1007-50 50 Zymo-Spin IV-HRC Spin Filters (Green Tops) C1010-50 50 Zymo-Spin IIC Columns C1011-50 50 Collection Tubes C1001-50 C1001-500 C1001-1000 50 500 1,000
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