Science Gateway > Protocols > Cell Biology Protocols - Table of Contents
Star Republic: Guide for Biologists

DNA labeling by nick translation (32P)

ChangBioscience.com
1. Cool a microcentrifuge tube on ice and mix the following on ice.

10X dNTP mix (- dCTP)
(0.2 mM each dATP, dGTP, dTTP, 500 mM Tris-HCl (pH 7.8)
50 mM MgCl2, 100 mM beta-mercaptoethanol, 100 mg/ml nuclease-free BSA)
2 ml
DNA (plasmid, BAC, YAC, etc.) 0.1-0.5 mg
10X Enzyme Mix
(0.5 U/ml DNA Polymerase I, 0.007 U/ml DNase I, 50 mM Tris-HCl (pH 7.5),
5 mM magnesium chloride, 0.1 mM phenylmethylsulfonyl fluoride,
50% (v/v) glycerol, 100 mg/ml nuclease-free BSA)
2 ml
a-32P-dCTP 5 ml
ddH20 to 20 ml

3. Mix thoroughly and centrifuge briefly. Incubate at 15 C for 2 hours.

4. Stop the reaction by placing the tubes in -20 C. Or stop the reaction by either adding 2 ml 0.5M EDTA (pH8.0) or heating to 75 C for 10 minutes.

5. Chill on ice.