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Star Republic: Guide for Biologists

DNA end-labeling

1. T4 polynucleotide kinase can be used to label double-stranded and single-stranded DNA or RNA at the 5'-ends.

2. g-32P-dATP is highly radioactive. Proper protection and waste disposal are required.

3. Prepare the following mixture:

ddH2O 4 ml
10X T4 polynucleotide kinase buffer 1 ml
DNA (0.1-1mg, 50-200 ng oligos) 2 ml
g-32P-dATP (5-10 gCi) 2 ml
T4 polynucleotide kinase 1 ml
Total 10 ml

4. Incubate at 37 C in a heat block reserved for radioactive reactions for 30-60 minutes.

5. Heat inactivate the T4 polynucleotide kinase at 65 C for 10 minutes.