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:
||up to 300 pmol of 5´ termini
|T4 PNK Reaction Buffer (10X)
|ATP (10 mM)
||1 µl (10 units)
||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).