Midi Plant Genomic DNA Purification Kit

Transcription

1 Midi Plant Genomic DNA Purification Kit Cat #:DP022MD/ DP022MD-50 Size:10/50 reactions Store at RT For research use only 1

2 Description: The Midi Plant Genomic DNA Purification Kit provides a rapid, simple and effective approach to isolate the genomic DNA from plant tissue. The process is based on a modified salt precipitation method, the procedures involve cell lysis at 65 C, precipitation of proteins, polysaccharides and secondary metabolites, removal of cell debris by filter paper, and DNA precipitation by isopropanol. Up to 1 g plant tissue can be processed and DNA purified with this kit is suitable for various applications, including PCR, restriction enzyme digestion, cloning, dot blot analysis, etc. Components of the Kit: DP022MD DP022MD Extraction Solution A* 160 ml 400 ml x 2 2. Extraction Solution B Powder concentrate 3.68 g (add 16 ml water) 18.4 g (add 80 ml water) 3. RNase A Powder** 110 mg 110 mg x 5 4. Precipitation Solution 60 ml 300 ml 5. Filter Paper 10 pcs 50 pcs * Upon storage at low temperature, Extraction Solution A may form SDS precipitate, which can be easily dissolved by incubating the bottle at 35 C. ** Store the lyophilized RNase A at -20 C, store all other kit components at room temperature(25~28 C). Materials to be supplied by the user: For tissue grinding: mortar and pestle. Liquid nitrogen Isopropanol 70% Ethanol 2

3 Before starting this procedure: To make Extraction Solution B Add sterile water to each tube of Extraction Solution B Powder Concentrate (16 ml for DP022MD, 80 ml for DP022MD-50) and mix by vortex to completely dissolve, incubate at 50 C water bath to assist dissolution. Aliquot to 5~10 tubes and store the solution at -20 C. Add 550 μl of sterile water to each tube of RNase A Powder and store at -20 C Check Extraction Solution A if any precipitate forms. If so, warm at 35 C to dissolve. Thaw the Extraction Solution B before grinding tissue. Preheat a water bath or heating block at 65 C, 60~65 C. Arrange 50 ml centrifugation tube (can use for >10,000 x g centrifugation) General Procedures: 1. Grind the plant tissue (250 mg~1 g) in liquid nitrogen to fine powder using mortar and pestle. Do not let sample to thaw. * For optimum yield and quality, do not use more sample than recommended amount. * Grind the tissue thoroughly. DNA in insufficiently ground tissue may be inaccessible to the reagents. * For intact DNA, use fresh plant tissue or those frozen (immediately after harvest) in liquid nitrogen and subsequently stored at -70 C. 2. Immediately transfer the powder to a 50 ml centrifuge tube. Add 15 ml Extraction Solution A, 1.5 ml Extraction Solution B and 50 μl RNase A solution, vortex vigorously for 5~10 sec. * Do not premix all these solutions. 3. Incubating at 65 C in water bath or heating block for 30 min. Mix by inverting the tube at frequent intervals. 4. Add 5.6 ml Precipitation Solution, mix by inverting the tube and 3

4 incubate on ice for 30 min. * The solution will become cloudy, because of precipitation of detergent, proteins, polysaccharides and secondary metabolites. 5. Centrifuge at top speed (12~14,000 x g) for 20 min at 4 C. 6. Prepare a filter paper in a funnel and carefully filter the supernatant into 50 ml centrifuge tube. 7. After filtration, add 12 ml isopropanol to the filtrate, mix well and incubate at -20 C for 30 min. * If DNA yield is lower than 2 μg, glycogen can be added(as a carrier) to a final 50 μg/ml. 8. Centrifuge at top speed for 20 min at 4 C. 9. Discard the supernatant carefully without disturbing the pellet. Wash the pellet with 10 ml 70% ethanol, and centrifuge at top speed for 10 min at 4 C. 10. 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. 11. Dry the pellet at heat oven (60~65 C) 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. 12. Resuspend the DNA pellet in 50~100 μl water or TE buffer. Store the eluted DNA at -20 C. 4

5 Troubleshooting Guide Comments and Suggestion Low or no DNA yield a) Too much sample b) Residual Ethanol contamination c) Lysate prepared incorrectly d) DNA is sheared or degraded Reduce the sample volume. Be sure to centrifuge at top speed for 10 min in step 9, or incubate at 60~65 C for 2 mins. The lysate must be handled gently after addition of isopropanol to prevent genomic DNA shearing. 1) Maintain a sterile environment while working to avoid DNase contamination. 2) Avoid repeated freezing and thawing of DNA samples 5

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