Non-radioactive phosphorylation with T4 PNK 3' phosphatase minus (M0236)
New England Biolabs
Published: 2022-04-12 DOI: 10.17504/protocols.io.4rm7vzdrlx1w/v2
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Abstract
Protocol for non-radioactive phosphorylation with T4 PNK 3' phosphatase minus.
Before start
Steps
1.
ATP is not supplied1X 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).
Set up the following reaction in a microcentrifuge tube On ice:
| A | B |
|---|---|
| Reagent | Volume |
| 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 |
Note
2.
Incubate at 37°C for 0h 30m 0s.
3.
Heat inactivate by incubating at 65°C for 0h 20m 0s.
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