Protocol for Dephosphorylation of 5´-ends of DNA using rSAP in Restriction Enzyme Reaction (M0371) also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 

  1. Digest 1–5 μg of plasmid DNA in a 20 μl reaction as follows:
    DNA > 1 µl
    Restriction Enzyme Buffer (10X) 2 µl
    Restriction Endonuclease 1 µl
    H2O, purified to 20 µl

    Note: Scale larger reaction volumes proportionally.

  2. Incubate at 37°C for 60 minutes or follow manufacturer’s recommendations.

  3. Add 1 unit of rSAP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and incubate at 37°C for 30–60 minutes. Visit our free tool NEBioCalculator to calculate pmol of ends in your reaction.

  4. Stop reaction by heat-inactivation of rSAP and restriction enzyme (follow manufacturer's recommendations).
Note: If restriction enzyme cannot be heat-inactivated, DNA purification is required before ligation.