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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)

    1. Set-up the following reaction in a microcentrifuge tube on ice:
    DNA up to 300 pmol of 5´ termini
    T4 PNK Reaction Buffer (10X)
    5 µl
    ATP (10 mM)
    5 µl
    T4 PNK 1 µl (10 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.

    • 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).