5’ overhangs can be end-labeled radioactively with T4 PolyNucleotide Kinase (PNK) , or filled in using the Klenow fragment with labeled dNTPs:
-1ul T4 PNK
-1ul 32P ATP (3,000Ci/mmol, 5mCi/ml)
-2ul 10x T4 PNK buffer
-1 or 2ul DNA ladder (1ug)
- Add distilled water to a reaction volume of 20ul.
- Incubate for 30 minutes at 37°C.
- Run the samples for 50 to 60 minutes at 100V in TBE buffer in a 4-20% acrylamide gel (10cm x 10cm). A 20 minutes exposure gives very readable signals. The signal strength is about twice that signal when ADP is added to 100uM.