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INSTRUCTION MANUAL ZR DNA-Card Extraction Kit Catalog No. D6040 Highlights Get the DNA out! Simple and efficient procedure for the purification of DNA from samples collected onto Guthrie, FTA, and other storage papers (cards). State-of-the-art, ultra-high density BashingBeads are fracture resistant, chemically inert, and can be used with any bead mill, disrupter, or vortex that can accommodate standard 2 ml tubes. Eluted DNA is high quality and inhibitor-free making it ideal for PCR and genotyping. Contents Product Contents & Specifications.. 1 Product Description..... 2 Protocol 3 Ordering Information.. 4 Xpedition Sample Prep Technologies... 5 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 are not satisfied with this product please call 1-888-882-9682. Product Contents ZR DNA-Card Extraction Kit (Kit Size) D6040 ( Preps.) Storage Temperature Proteinase K & Storage Buffer* 2x 5 mg 20 C (after mixing) 2X Digestion Buffer** 20 ml Room Temp. Lysis Solution 40 ml Room Temp. DNA Isolation Buffer ml Room Temp. DNA Wash Buffer 6 ml Room Temp. DNA Elution Buffer 10 ml Room Temp. ZR BashingBead Tubes (2.0 mm) Room Temp. Zymo-Spin IC Columns Room Temp. Collection 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. * The Proteinase K is stable as shipped. Add 260 µl Proteinase K Storage Buffer to each Proteinase K tube prior to use. The final concentration of Proteinase K after the addition of Proteinase K Storage Buffer is ~20 mg/ml. ** The 2X Digestion Buffer may have formed a precipitate during shipping. If this is the case, incubate at 37 o C to solubilize. Specifications Note: DNA yield and integrity may vary significantly depending on the sample input and quality, respectively. Sample Sources Blood, saliva, and cells collected onto Guthrie, FTA, and other storage papers (cards). Typically, portions of a card are sectioned, minced, or punched (1.2 7.0 mm) for input and processing with the kit. If needed, multiple punches can be input into a lysis tube. DNA Purity High quality DNA for PCR, endonuclease digestion, Southern blotting, bisulfite conversion/methylation detection, sequencing, genotyping, etc., is eluted with DNA Elution Buffer or water. (A260/A280 1.8) DNA Size Capable of recovering genomic and mitochondrial DNA sized fragments from bp to 40 kb. If present, parasitic, microbial, and viral DNA will also be recovered. DNA Yield The DNA binding capacity of the column is ~5 µg. Depending on the sample source and age of the archived sample, DNA yields will vary. DNA can be eluted from the column using 10 µl DNA Elution Buffer or water. Product Detergent Tolerance 5% Triton X-, 5% Tween-20, 5% Sarkosyl, 1% SDS. Equipment Homogenizer (bead beater), water bath or heat block (55 o C), and microcentrifuge. 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. FTA is a registered trademark of GE Healthcare. FastPrep is a registered trademark of MP Biomedicals.

Page 2 Product Description DNA can be notoriously difficult to elute from some cellulose-based storage papers (cards) 1,2. The ZR DNA-Card Extraction Kit is designed for simple and rapid purification of inhibitor-free, PCR-quality DNA from blood, saliva, and cells collected onto Guthrie, FTA, and other storage papers (cards). The procedure is easy and can be completed in about 30 minutes: card punches are added directly to a ZR BashingBead Lysis Tube and thoroughly homogenized by bead beating (e.g., Xpedition Sample Processor, FastPrep -24, or similar). Following Proteinase K digestion, the DNA is purified using innovative column technology (see schematic of procedure below). Eluted DNA is ideal for PCR, genotyping, etc. Punch References: 1. Sigurdson AJ, et al., Cancer Epidemiol Biomarkers Prev 6 Feb;15(2)385-8 Figure 1. DNA can be effectively purified from blood spotted onto Guthrie and FTA cards using the ZR DNA-Card Extraction Kit. Briefly, two punches (3.0 mm) from blood spotted onto a Guthrie (C1) and FTA (D1) card (>1 yr. old) was used for processing with the kit. Eluted DNA was analyzed with a TapeStation (Agilent) shown in the image above. A1 is a molecular weight marker and B1 input DNA from a fresh blood preparation. 2. Milne E, et al., Cancer Epidemiol Biomarkers Prev 6;15:816-819

Page 3 For Technical Assistance, please contact 1-888-882-9682 or E-mail tech@zymoresearch.com. Reagent Preparation Add 260 µl Proteinase K Storage Buffer to each Proteinase K tube prior to use. The final concentration of Proteinase K after the addition of Proteinase K Storage Buffer is ~20 mg/ml. Add 24 ml % ethanol (26 ml 95% ethanol) to the 6 ml DNA Wash Buffer concentrate. Protocol Perform all centrifugation steps at 10,000 x g. 1. Add card samples (punches) to a ZR BashingBead Lysis Tube. Add 400 µl Lysis Solution to the tube. Alternatively, a vortex may be used; however, processing times may need to be increased to 5-10 minutes. 2. Secure lysis tube in a bead beater fitted with a 2 ml tube holder assembly and process at maximum speed. Note: Processing times may be as little as 40 seconds when using high-speed disrupters (e.g., FastPrep -24, or similar). See manufacturer s literature for operating instructions. 3. Centrifuge the ZR BashingBead Lysis Tube for 1 minute. 4. To the lysate in the ZR BashingBead Lysis Tube, add: 2X Digestion Buffer 390 µl Proteinase K 10 µl 5. Mix and then incubate the tube at 55 o C for 15-30 minutes. 6. Centrifuge the ZR BashingBead Lysis Tube for 1 minute. Transfer 400 µl supernatant to a microcentrifuge tube. 7. Add 800 µl DNA Isolation Buffer to the tube and mix thoroughly. The column capacity is 800 µl. 8. Transfer 800 µl of the mixture to a Zymo-Spin IC Column in a Collection Tube. Centrifuge for 1 minute. Discard flow through and repeat until the entire volume has passed through the column. 9. Add µl of DNA Wash Buffer to the spin column. Centrifuge for one minute. Repeat this wash step. Elution of DNA from the column is dependent on ph and temperature. If water is used, ensure the ph is >6.0. Also, the total yield may be improved by eluting the DNA with Elution Buffer or water pre-equilibrated to 60-70 o C or by performing and pooling sequential elutions. 10. Transfer the spin column to a clean microcentrifuge tube. Add 10 µl DNA Elution Buffer or water to the spin column. Incubate 2-5 minutes at room temperature, then centrifuge at top speed for 30 seconds to elute the DNA. The eluted DNA can be used immediately for molecular based applications or stored -20ºC for future use.

Page 4 Ordering Information Product Description Catalog No. Kit Size ZR DNA-Card Extraction Kit D6040 preps. For Individual Sale Catalog No. Amount Proteinase K & Storage Buffer 2X Digestion Buffer Lysis Solution D3001-2-5 D3001-2-20 D30-1-5 D30-1-20 D6001-3-40 D6001-3-1 5 mg set 20 mg set 5 ml 20 ml 40 ml 1 ml DNA Isolation Buffer D4010-1- ml DNA Wash Buffer DNA Elution Buffer D4003-2-6 D4003-2-24 D3004-4-4 D3004-4-10 D3004-4- 6 ml 24 ml 4 ml 10 ml ml ZR BashingBead Lysis Tubes (2.0 mm) S6003- tubes Zymo-Spin IC Columns Collection Tubes C4- C4-2 C1- C1-0 C1-0 columns 2 columns tubes 0 tubes 1,000 tubes

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Page 6 Popular Products from Zymo Research Product DNA Clean & Concentrator -5 DNA Clean & Concentrator -25 ZR-96 DNA Clean & Concentrator -5 Genomic DNA Clean & Concentrator Zymoclean Gel DNA Recovery Kit ZR-96 Zymoclean Gel DNA Recovery Kit Zymoclean Large Fragment DNA Recovery Kit OneStep PCR Inhibitor Removal Kit Zyppy Plasmid Miniprep Kit Zyppy -96 Plasmid Miniprep Zyppy Plasmid Midiprep Kit ZR Plasmid MiniPrep Classic Quick-gDNA MiniPrep ZR-96 Quick-gDNA ZR-Genomic DNA - Tissue MiniPrep Environmental DNA Purification Kits Description Fragment DNA Purification Clean and concentrate up to 5µg DNA into 6 µl elution volume in as little as 2 minutes with no wash residue carryover. Clean & concentrate 25 µg of DNA into 25 µl elution volume in as little as 2 minutes with no wash residue carryover. Quick (15 minute), high-output recovery of up to 5 µg pure DNA into 10-15 µl minimum elution volume allows for highly concentrated DNA. Quick (5 minute) clean-up of up to 10 µg high molecular weight DNA ( kb) from any enzymatic reaction or impure preparation without precipitations. Purify DNA from high and low-melting agarose gels in minutes High-throughput DNA purification from high and low-melting agarose gels. Purify high molecular weight DNA ( kb) from high and low-melting agarose gels in minutes Fast, one step procedure for removal of PCR inhibitors such as polyphenolics, humic/fulvic acids, melanin, etc. for successful PCR and other downstream applications. Plasmid DNA Purification Pellet-Free plasmid DNA purification in less than 10 minutes. Recover up to 25 µg DNA in as low as 30 µl. The fastest and simplest high-throughput method for plasmid purification. Pellet-Free plasmid DNA purification in 15 minutes in a 1 µl minimum elution volume). Plasmid DNA purification in minutes: (alkaline lysis/spin column format for low 30 µl elution volume). Genomic DNA Purification Easy purification of genomic DNA from whole blood, plasma, serum, body fluids, buffy coat, lymphocytes, tissue, swabs or cultured cells in as little as 15 minutes without the use of Proteinase K or organic denaturants. Simple, high throughput (96-well) purification of DNA from whole blood, plasma, serum, body fluids, buffy coat, lymphocytes, tissue, swabs, or cultured cells in about 30 minutes. For high quality DNA purification from solid tissues (e.g., tail snips, ear punches, adipose tissue, etc.), body fluids, cultured cells, buccal cells, FFPE tissues, hair, and other biological sources using Proteinase K and Fast. Unique BashingBead technology allows isolation of DNA from samples refractory to conventional lysis procedures including tough-to-lyse tissues, soil samples, feces, plants, seeds, insects, bacteria, yeast, filamentous fungi, unicellular and filamentous algae, and protozoa Please visit our website to see our complete line-up of products. Kit Size (Preps) 25 25 400 8x96 25 400 10x96. Catalog No. (Format) D4003 (uncapped) D4004 (uncapped) D4013 (capped) D4014 (capped) D4005 (uncapped) D4006 (uncapped) D4033 (capped) D4034 (capped) D4023 D4024 D4010 (capped) D4011 (capped) D4001 (uncapped) D4002 (uncapped) D4007 (capped) D4008 (capped) D4021 D4022 D4045 (capped) D4046 (capped) D6030 D6035 D4036 D4019 D4020 D4041 D4042 D4043 D4025 D4026 D4036 D4019 D4020 D3006 (uncapped) D3007 (uncapped) D3024 (capped) D3025 (capped) D3010 D3011 D3012 D30 D3051 Visit website for a comprehensive list