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  • Non-radioactive phosphorylation with T4 PNK or T4 PNK 3' phosphatase minus

    Introduction

    For non-radioactive phosphorylation, use up to 300 pmol of 5' termini in a 50 µl reaction containing 1X T4 Polynucleotide Kinase Buffer, 1 mM ATP and 10 units of T4 Polynucleotide Kinase. Incubate at 37°C for 30 minutes. 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).