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Star Republic: Guide for Biologists

Incoporation efficiency --- filter binding assay

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

2. In duplicate, spot 3 Ál of the diluted sample on Whatman DE 81 circular filters.

3. Dry the filters briefly under a heat lamp. One filter will be used directly for the determination of total cpm in the sample.

4. Wash the other filter in 50 ml of 0.5 M sodium phosphate (pH 6.8) for 5 minutes to remove the unincorporated dNTPs.

5. Repeat wash for 5 minutes.

6. Dry the washed filter under a heat lamp.

7. Add the appropriate amount of scintillation fluid to each filter and count in a scintillation counter.