1. Add 300-450 mg dried tissue (finely ground) or 6001,200 mg frozen tissue (which may be finely ground with a mortar and pestle in liquid nitrogen), or 6001,200 mg fresh tissue to a 50 ml tube. Work quickly and keep tissue cold to minimize DNase activity. Note: it may be necessary to vary the amount of starting material depending upon the species, age, tissue preparation, and genome size.
2. Add 18 ml cell lysis solution to the leaf tissue. For dried tissue, cap the tubes and vortex 13 sec to wet the tissue. For unground tissue, homogenize using 3050 strokes with a tube pestle.
3. Incubate cell lysate at 65C for 60 min. After 30 and 60 min, invert capped tube 10 times.
RNase Treatment (Optional)
1. Add 90 l RNase A solution to the cell lysate.
2. Mix the sample by inverting the capped tube 25 times and incubate at 37C for 15 min.
1. Cool sample to room temperature.
2. Add 6 ml protein precipitation solution to the lysate.
3. Cap the samples and vortex vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate. Alternatively, invert a rack containing the samples 150 times (approximately 2 min) to mix the protein p recipitation solution uniformly with the cell lysate. For species with high polysaccharide content, it may be necessary to incubate sample on ice for 515 min.
4. Centrifuge at 3,000 x g for 10 min. The precipitated proteins should form a tight, green pe llet. The supernatant may range in appearance from a transparent brown to green, depending on the sample. If the pellet is not tight, repeat step 3, incubate on ice for 5 min, then repeat step 4.
1. Leaving behind the precipitated protein pellet, pour the supernatant containing the DNA into a 50 ml centrifuge tube that contains 18 ml 100% isopropanol (2-propanol).
2. Mix the sample by inverting the capped tube gently 50 times.
3. Centrifuge at 3,000 x g for 5 min. The DNA will be visible as a pellet that ranges in color from off-white to dark green, depending on particulates that cosediment with the DNA.
4. Pour off supernatant and drain tube briefly on clean absorbent paper. Add 18 ml 70% ethanol and invert the capped tube several times to wash the DNA pellet.
5. Centrifuge at 3,000 x g for 5 min. Carefully pour off the ethanol. Pellet may be loose, so pour slowly and watch the pellet to ensure that it stays in the tube.
6. Invert and drain the tube on clean absorbent paper and allow to air-dry 15 min.
1. Add 1 ml DNA hydration solution.
2. Rehydrate DNA by incubating sample 1 hr at 65C, or a l t e rn a t i v e l y, overnight at room temperature. Tap tube periodically to aid in dispersing the DNA. Transfer DNA sample to a clean 1.5 ml tube for storage.
3. If particulates are present in the rehydrated DNA sample, centrifuge at 13,00016,000 x g for 510 min and transfer supernatant into a clean 1.5 ml tube. Store DNA at 28C.
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