Protocol for Dephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289)

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Protocol

1. 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**

   
   
   
2. Incubate at 37°C for 30 minutes.
   
   
3. 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