Protocol for Dephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289) also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 


  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 phosphate 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