Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (NEB #M0371) 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*
    rCutSmart™ Buffer (10X) 2 µl
    rSAP 1 unit
    Nuclease-free water 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 DNA in Restriction Enzyme Reaction

  • The phosphatase 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