Non-radioactive Phosphorylation with T4 PNK or T4 PNK (3´ phosphatase minus) 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).