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Star Republic: Guide for Biologists

DNA random labeling with Biotin/Digoxigenin

Virtualab Protocols (Chang Bioscience)
1. Add 20-100 ng of template DNA and ddH2O to a final volume of 32 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, dNTP, Biotin-16-dUTP or Digoxigenin-11-dUTP, random hexamer primers, and Klenow enzyme.

4. Make the following mix:

Denatured DNA (20-100 ng) 32 ml
5X Labeling Buffer
(containing random primers, dNTP, Biotin-16-dUTP or
Digoxigenin-11-dUTP, and Klenow enzyme)
8 ml

Final concentration of dNTP is 0.1 mM dTTP, 0.1 mM Biotin-16-dUTP or Digoxigenin-11-dUTP, 0.2 mM dATP, 0.2 mM dCTP, and 0.2 mM dGTP.

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 tube cap lock.