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  • Radioactive Labeling with T4 PNK or T4 PNK (3´ phosphatase minus)

    Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community. This is available for both the Radioactive Labeling with T4 PNK and the Radioactive Labeling with T4 PNK (3´ phosphatase minus). 

    1. Set-up the following reaction:
    DNA Up to 50 pmol of 5´ termini
    T4 PNK Reaction Buffer (10X)
    5 µl
    ATP (10 mM)
    50 pmol of [γ-32P] ATP
    T4 PNK 2 µl (20 units)
    Nuclease-free Water up to 50 µl

    2. Incubate at 37°C for 30 minutes.
    3. Heat inactivate by incubating at 65°C for 20 minutes.

    Note: [33P] ATP may be substituted for [32P] ATP.