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Star Republic: Guide for Biologists

Incoporation efficiency --- TCA precipiation

1. Dilute 1 Ál of the sample 1:100 in 0.2 M EDTA (pH 8.0).

2. Spot 3 Ál of the diluted sample on a glass fiber (Whatman GF/C glass-fiber disc) or nitrocellulose filter for determination of total cpm in the sample.

3. Transfer 3 Ál of the same dilution to a tube. Add 100 Ál of Salmon sperm DNA (50 mg/ml). Mix well.

4. Add 1.0 ml of 10% TCA (ice-cold). Leave on ice for 15 minutes.

5. Collect the precipitated DNA by vacuum filtration onto a glass fiber or nitrocellulose filter.

6. Wash the filter 6 times with 5 ml of cold 10%TCA and once with 5 ml of 95% ethanol.

7. Dry both filters in the air or under a lamp.

8. Add an appropriate amount of scintillation fluid to each filter and count using a scintillation counter.