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DNA Isolation From 300 to 450 mg Dried or 600 to 1,200 mg Fresh Leaf Tissue

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.

DNA Precipitation
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.

DNA Hydration
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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