( 4. Centrifuge at 1300016000 x g fo...)DNA Isolation From 50 to 20 mg of Paraffin-Embedded Tissue

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.

DNA Precipitation
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.

DNA Hydration
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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