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Star Republic: Guide for Biologists

DNA random labeling with 32P-dCTP

1. a-32P-dCTP is highly radio active, proper protection and waste disposal are required.

2. Add 10-25 ng of template DNA and ddH2O to a final volume of 11 ml in a microcentrifuge tube.

3. Denature the DNA sample by heating to 95-100 C for 5 minutes. Chill on ice.

For use with DNA labeling kits. The 5X labeling mix solution contains the reaction buffer, random hexamer primers, and Klenow enzyme.

4. Make the following mix:

Denatured DNA (10-25 ng) 11 ml
5X Labeling Buffer (containing random primers and Klenow enzyme) 4 ml
a-32P-dCTP 5 ml

For use with Klenow. The reaction buffer, random hexamer primers, and Klenow enzyme need to be added separately.

4a. Make the following mix:

Denatured DNA (10-25 ng) 9 ml
10X Klenow Reaction Buffer 2 ml
Random hexamer or octomer primer ( ~750 ng, 30 pmol) 2 ml
Klenow ( 5 units/ml ) 1 ml
a-32P-dCTP 5 ml

5. Incubate at 37 C for 10-60 minutes.

6. Stop the reaction by adding 1 ml of 0.5 M EDTA or by heating heating to 95-100 C for a couple of minutes.

7. Immediately before use in hybridization, denature the labeled DNA by heating to 95-100 C for 5 minutes and then put on ice. To avoid poping up of the microcentrifuge tube, puncture a small hole on the cap using a needle or use a microcentrifuge cap locker.