1. Place 510 mg (0.0050.010 g) of finely minced tissue in a 1.5 ml capped tube. Add 300 l xylene and incubate 5 min with constant mixing at room temperature.
2. Centrifuge at 13,00016,000 x g for 13 min to pellet the tissue. Discard the xylene.
3. Repeat steps 1 and 2 twice (for a total of 3 xylene washes).
4. Add 300 l of 100% ethanol to the tube and incubate 5 min with constant mixing at room temperature.
5. Centrifuge at 13,000-16,000 x g for 1-3 min to pellet the tissue. Discard the ethanol.
6. Repeat steps 4 and 5 (for a total of 2 ethanol washes).
1. Add 300 l cell lysis solution, and homogenize using 3050 strokes with a microfuge-tube pestle.
2. Incubate lysate at 65C for 1560 min.
3. 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 until tissue particulates have dissolved (3 hr to overnight). 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 A-tre a t e d cell lysate.
3. Vortex capped tube vigorously at high speed for 20 sec to mix the protein precipitation solution uniformly with the cell lysate.
4. Centrifuge at 13,00016,000 x g for 3 min. The precipitated proteins will form a tight pellet. If the protein pellet is not visible, repeat step 2 followed by incubation on ice for 5 min, then repeat step 3.
1. Leaving behind the precipitated protein pellet, pour the supernatant that contains the DNA into a clean 1.5 ml microfuge tube containing 300 l 100% isopropanol (2-propanol). If DNA yield is expected to be low (<1 g), add 0.5 l glycogen solution (20 mg/ml) to the isopropanol.
2. Cap the tube and mix by inverting gently 50 times.
3. Centrifuge at 13,00016,000 x g for 5 min.
4. Pour off supernatant and drain tube 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 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 for 15 min.
1. Add 20 l DNA hydration solution (20 l will give a concentration of 100 ng/l if the total yield is 2 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.
3. Store DNA at 28C.
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