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)
5X Labeling Buffer
(containing random primers, dNTP, Biotin-16-dUTP or
Digoxigenin-11-dUTP, and Klenow enzyme)
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.