Protocol for Dephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289)
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- Prepare a 20 μl reaction as follows:
DNA 1 pmol of DNA ends* Antarctic Phosphatase Reaction Buffer (10X) 2 μl Antarctic Phosphatase 5 units H2O, purified to 20 μl**
- Incubate at 37°C for 30 minutes.
- Stop reaction by heat-inactivation at 80°C for 2 minutes.
* Note: 1 pmol of DNA ends is about 1 μg of a 3 kb plasmid.
** Scale larger reaction volumes proportionally.
Dephosphorylation of 5´-ends of DNA in Restriction Enzyme Reaction
- The phosphatase can be added directly into the digestion reaction during or after DNA digestion
- Antarctic Phosphatase is active in all NEB restriction enzyme buffers only when supplemented with Antarctic Phosphatase Reaction Buffer, which provides Zn2+ required for enzyme activity
- The restriction enzyme should be heat inactivated at the same time as the phosphatase after digest and dephosphorylation
- If restriction enzyme cannot be heat inactivated, DNA purification is required before ligation