1. Dissect tissue sample quickly and freeze in liquid nitrogen. Store at -70 to -80C. Fresh tissue may also be used. Work very quickly and keep tissue on ice at all times including when tissue is weighed.
2. Add 510 mg (0.0050.01 g) frozen ground tissue or fresh tissue to a 1.5 ml centrifuge tube containing 300 l cell lysis solution, remove from ice, and homogenize thoroughly using a m i c rofuge tube pestle. Place sample back on ice until next step.
3. Incubate lysate at 65C for 1560 min. Alternatively, if maximum yield is required, 1.5 l proteinase K solution (20 mg/ml) may be added to the lysate. Mix by inverting capped tube 25 times and incubate at 55C for 3 hr to overnight, until tissue particulates have dissolved. If possible, invert tube periodically during the incubation.
1. Add 1.5 l RNase A solution (4 mg/ml) to the cell lysate.
2. Mix the sample by inverting the capped tube 25 times and incubate at 37C for 1560 min.
1. Cool sample to room temperature.
2. Add 100 l protein precipitation solution to the RNase Atreated cell lysate.
3. Vo rtex vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate. For samples with high polysaccharide content, incubate on ice for 515 min.
4. Centrifuge at 13,00016,000 x g for 3 min. For samples with high polysaccharide content, centrifugation at 4C may be required. The precipitated proteins should form a tight pellet. If the protein pellet is not tight, repeat step 3 and include the incubation on ice, then repeat step 4.
1. Pour the supernatant containing the DNA (leaving behind the precipitated protein pellet) into a 1.5 ml microfuge tube containing 300 l 100% isopropanol (2-propanol).
2. Cap the tube and mix the sample by inverting gently 50 times.
3. Centrifuge at 13,00016,000 x g for 1 min; the DNA will be visible as a small white pellet.
4. Pour off supernatant and drain tube briefly on clean absorbent paper. Add 300 l 70% ethanol and invert capped tube several times to wash the DNA pellet.
5. Centrifuge at 13,00016,000 x g for 1 min. Carefully pour off the ethanol. Pellet may be loose, so pour slowly and watch pellet to ensure it stays in the tube.
6. Invert and drain the tube on clean absorbent paper and allow to air-dry for 15 min.
1. Add 50 l DNA hydration solution (50 l will give a concentration of 100 g/ml if the total yield is 5 g DNA).
2. Allow DNA to rehydrate overnight at room temperature. Alternatively, heat at 65C for 1 hr. Tap tube periodically to aid in dispersing the DNA. Store DNA at 28C.
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