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  • Non-radioactive phosphorylation with T4 PNK or 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).