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  • 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 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