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DNA Quantification - TCA Precipitation

Jeff Lawrence (Z01)

1. Dilute radioactive sample to a 100 ml volume

2. Spot 5 ml of the sample onto the center of a 2.4 cm Whatman GF/C glass-fiber disc.

3. Mix 5 ml of the sample with 100 ml Salmon sperm DNA (50 mg in 20 mM EDTA).

4. Add 5 mL ice cold 10% Trichloroacetic acid (TCA). Mix well and incubate on ice for 15 min.

5. Filter the solution through a separate GF/C glass-fiber disc.

6. Wash the filter 6 times with 5 mL ice-cold 10% TCA.

7. Wash filter once with 5 mL 95% Ethanol.

8. Dry both filter under a heat lamp.

9. When dry, count each in a scintillation counter.

10. The first filter measures total radioactivity. The second filter measures radioactivity incorporated into DNA fragments greater than 20 nucleotides in length.