Wednesday, October 08, 2003

Description
Typical 5'-kinase labeling reactions included the DNA to be labeled, [[gamma]]-32-P-dATP, T4 polynucleotide kinase, and buffer (1). After incubation at 37degC, reactions are heat inactivated by incubation at 80degC. Portions of the reactions are mixed with gel loading dye and loaded into a well of a polyacrylamide gel and electrophoresed. The gel percentage and electrophoresis conditions varied depending on the sizes of the DNA molecules of interest. After electrophoresis, the gel is dried and exposed to x-ray film, as discussed below for radiolabeled DNA sequencing.

Primary Author
Bruce A. Roe ( broe@ou.edu )

Affiliation
University of Oklahoma , United States

Co-Author(s)
Judy S. Crabtree
Akbar S. Khan

Procedure
1. Add the following reagents to a 0.5 ml microcentrifuge tube, in the order listed:

sterile ddH2O: q.s
10X kinase buffer: 1 ul
DNA: x ul
[[gamma]]-[32-P]-dATP: 10 uCi
T4 polynucleotide kinase: 1 ul (3U/ul)
Total: 10 ul


[[gamma]]-[32-P]-dATP (35020) ICN and T4 polynucleotide kinase (70031) from United States Biochemicals.

2. Incubate at 37degC for 30-60 minutes.


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