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End-labeling (N3200)

Introduction 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) Protocol 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.