DNA Quantification - TCA Precipitation
Jeff Lawrence (Z01)
1. Dilute radioactive sample to a 100 µL volume
2. Spot 5 µL of the sample onto the center of a 2.4 cm Whatman GF/C glass-fiber disc.
3. Mix 5 µL of the sample with 100 µL 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.