Dephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289)
New England Biolabs
Published: 2022-02-14 DOI: 10.17504/protocols.io.bd2xi8fn
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Abstract
This is the protocol for dephosphorylation of 5'-ends of DNA using AnP (Antarctic Phosphatase - M0289).
Before start
Steps
1.
Prepare a 20µL reaction as follows:
| A | B |
|---|---|
| COMPONENT | AMOUNT |
| DNA | 1 pmol of DNA ends* |
| Antarctic Phosphatase Reaction Buffer (10X) | 2 μl |
| Antarctic Phosphatase | 5 units |
| H2O, purified | to 20 μl** |
- Note: 1 pmol of DNA ends is about 1 μg of a 3 kb plasmid.** Scale larger reaction volumes proportionally.
2.
Incubate at 37°C for 0h 30m 0s.
3.
Stop reaction by heat-inactivation at 80°C for 0h 2m 0s.
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