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