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  • End-labeling Protocol

    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

    1. Add distilled water to a reaction volume of 20ul.
    2. Incubate for 30 minutes at 37°C.
    3. 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.