Easy Tissue & Cell Genomic DNA Purification Kit Cat. #:DP021E/ DP021E-150 Size:50/150 reactions Store at RT For research use only 1
Description: The Easy Tissue & Cell Genomic DNA Purification Kit is ideal for isolating genomic DNA from various animal tissues, cultured cells, and bacteria. This kit does not require organic solvents such as phenol and chloroform, it is safe and user-friendly. The Easy Tissue & Cell Genomic DNA Purification Kit is designed for isolating DNA from 1~25 mg tissue samples, up to 1 x 10 7 cultured tissue cells, or up to 4 x 10 9 bacterial cells. Typical yield ranges from 30~80 μg of DNA, depending upon the sample volume and sample type. The purified DNA is suitable for various applications, including PCR, and restriction enzyme digestion. Components of the Kit: DP021E DP021E-150 1. Extraction Solution* 35 ml 105 ml 2. Proteinase K Powder** 22 mg 22 mg x 3 3. RNase A Powder** 44mg 44 mg x 3 4. Protein Precipitation Solution 12 ml 36 ml * Upon storage at low temperature, Extraction Solution may form SDS precipitate, which can be easily dissolved by incubating the bottle at 35 C. ** Store the lyophilized Proteinase K & RNase A at -20 C, store all other reagents and kit components at RT(15~30 C). ** Proteinase K may turn yellow after long term storage. This is a normal phenomenon. Equipment and reagents to be supplied by the user: Mortar and pestle Trypsin (for adherent tissue culture cells only) 1
180ul Lysozyme solution (for gram-positive bacteria strains) [4 mg lysozyme powder in 20 mm Tris-HCl (ph 8.0), 2 mm EDTA, 1.2% Triton X-100]. Isopropanol 70 % Ethanol Things to do before starting Dissolve Proteinase K in 1.1 ml sterile H 2 O and store at -20 C. If RNA-free genomic DNA is desired, prepare RNase A solution by dissolving RNase A powder in 220 μl sterile H 2 O and store at -20 C Preheat water bath or heat block at 56 C or 70 C, respectively. General Procedures: * Please follow suggested sample amount to avoid lowering DNA quality or DNA extraction becoming too viscous to handle. 1. A. Tissue culture cells (< 1 x 10 7 ): (1) Harvest the cells (for adherent cells, trypsinize cells before harvesting) and transfer to 1.5 ml microcentrifuge tube. (2) Centrifuge at 14,000 xg for 10 sec to pellet the cells and carefully remove most of the supernatant. Leave approximately 50 µl of supernatant with the pellet. (3) Resuspend the pellet completely by pipetting. Add 600 µl of Extraction Solution and vortex thoroughly. Proceed to Step 2. B. Animal or insect tissue (< 25 mg), spleen (< 10 mg), mouse tail (< 1.5 cm): There are two methods available for tissue treatments: (1) Cut the tissue into small pieces and transfer the tissue to 1.5 ml microcentrifuge tube. Add 600 µl of Extraction Solution, and proceed 2
to Step 2. (Tissue lysis will be more time consuming as the tissue is not homogenized). (2) Homogenize the tissue in liquid nitrogen and transfer the homogenate to 1.5 ml microcentrifuge tube. Add 600 µl of Extraction Solution and vortex thoroughly. Proceed to Step 2. C. Bacteria: (1) Gram-negative bacteria: Centrifuge cells (< 4 x 10 9 ) for 2 min, carefully remove the supernatant and resuspend bacterial pellet in 600 µl Extraction Solution. Vortex thoroughly, and proceed to Step 2. (2) Gram-positive bacteria: Centrifuge cell (< 4 x 10 9 ) for 2 min, carefully remove the supernatant and resuspend the pellet in 180 μl of Lysozyme solution. Incubate at 37 C for 30 min and add 600 μl Extraction Solution. Proceed to Step 2. D. Yeast cell: Please refer to appendix on page 5. 2. Add 20 µl of proteinase K stock solution to the microcentrifuge tube, mix by vortexing. * Do not premix Extraction Buffer and Proteinase K solution before use to prevent proteinase K from undergoing self-digestion without substrate. 3. Incubate the samples at 56 C in water bath or incubator for 1~3 hours or longer until complete lysis of tissue (time varies depending on sample size), vortex 5~10 sec at frequent invertals during incubation. * If the sample mixture is too viscous to pipette, incubate overnight to ensure complete lysis. Sample Time Cultured cells from tissue (< 5 X 10 6 ) 0.5-1 hours 3
Animal or insect tissue (< 25 mg), for spleen (< 10 mg) Bacteria Yeast cell 1-3 hours 0.5-1 hours 0.5-1 hours 4. Optional: If RNA-free DNA is desired, add 4 µl RNaseA solution to the sample and incubate at 37 C for 15~30 minutes. 5. Add 200 µl of Protein Precipitation Solution to reaction, vortex thoroughly. 6. Centrifuge the sample at top speed(12~14000x g) for 3 min at RT. * If pellet does not form, incubate on ice for 5 min and centrifuge again. 7. Transfer the supernatant without disturbing the pellet into a new microcentrifuge tube. 8. Add 600 µl isopropanol, mix thoroughly by inverting the tube several times until the DNA stringy precipitate forms well. * If DNA content is limited, glycogen can be added to a final concentration of 50 μg /ml before adding isopropanol. 9. Centrifuge the sample at top speed for 1 min at RT. 10. Discard the supernatant carefully without disturbing the pellet. Wash the pellet with 1 ml 70% ethanol. 11. Centrifuge at top speed for 1 min at room temperature to pellet the DNA. 12. Discard the supernatant carefully, spin briefly and pipet out all the ethanol. * Remove as much supernatant as possible, which will fasten and control the DNA drying time 13. Dry the pellet at room temperature for 5~10 min or 50 C incubator for 2 min. * Do not incubate too long to over-dry the pellet, which will make DNA hard to dissolve. * Do not use SpeedVac, which will make DNA hard to dissolve. 14. Resuspend the DNA pellet in 100~200 μl water or TE buffer. Store the eluted 4
DNA at -20 C. * Incubate the tube at 65 C for a few minutes and flick the tube occasionally to help DNA dissolve completely into solution. Appendix: protocol for isolation of genomic DNA from yeast Additional reagents required: Sorbitol buffer (fresh prepared): 1 M sorbitol 100 mm EDTA 14 mm β-mercaptoethanol Lyticase (ICN Biomedical) or zymolyase (Zymo Research) Ex: yeast lytic enzyme 5g, add water 200μl(final conc.50u/μl),aliquot to 5~10 tubes, add 4μl per reaction. Procedures: 1. Spin the cells (< 5x 10 7 ) at 5000 x g for 10 min and carefully remove the supernatant. 2. Resuspend the pellet in 600 μl Sorbitol buffer, add about 200 U lyticase or zymolyase and incubate at 30 C for 30~60 min. * Incubation time may vary depending on cell numbers, species, and enzyme activity. See the guideline from enzyme supplier to monitor the incubation time. 3. Spin down the spheroplasts at 300 x g for 10 min. * The cells may be fragile after enzyme digestion, centrifuge with less g force. 4. Resuspend the spheroplasts in 200 μl Extraction Solution. 5. Continue to step 2 as per General Procedure on page 3. 5
Troubleshooting Guide Low or no DNA yield Comments and Suggestion a) Too much sample Reduce the sample volume. b) Residual Ethanol contamination Be sure to centrifuge at top speed for 1 min in Step 11. and RT for 5 ~10 min or 50 C incubator for 2 min. c) Lysate prepared incorrectly The lysate must be handled gently after addition of isopropanol to prevent genomic DNA shearing. d) DNA is sheared or degraded 1) Avoid extensive pipetting to facilitate lysis /homogenization or vortexing to prevent shearing of DNA. 2) Maintain a sterile environment while working to avoid DNase contamination. 3) Avoid repeated freezing and thawing of DNA samples 6
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