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  • Non-radioactive phosphorylation 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. It is available for both the Non-Radioactive Phosphorylation with T4 PNK and the Non-Radioactive Phosphorylation with T4 PNK 3' phosphatase minus

    Set-up the following reaction:
    DNA up to 300pmol of 5' termini
    10X T4PNK Reaction Buffer 5 µl
    10mM ATP 5 µl
    T4 PNK 1 µl (10 units)
    Water up to 50 µl

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

    Notes:
    • ATP is not supplied
    • 1X T4 DNA Ligase Buffer contains 1 mM ATP and can be substituted in non-radioactive phosphorylations (T4 Polynucleotide Kinase exhibits 100% activity in this buffer).