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  • Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (M0371)

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    1. Prepare a 20 μl reaction as follows:
      DNA 1 pmol of DNA ends*
      CutSmart® Buffer (10X) 2 µl
      rSAP 1 unit
      H2O, purified to 20 µl**

    2. Incubate at 37°C for 30 minutes.

    3. Stop reaction by heat-inactivation at 65°C for 5 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 in Restriction Enzyme Reaction

    • The phosphate can be added directly into the digestion reaction during or after DNA digestion
    • rSAP is active in all NEB restriction enzyme buffers
    • The restriction enzyme should be heat inactivated at the same time as the phosphatase after digest and dephosphorylation
    • If restriction enzyme(s) cannot be heat inactivated, DNA purification is required before ligation