Home Protocols Protocol for Dephosphorylation of 5´-ends of DNA using rSAP in Restriction Enzyme Reaction (M0371)

Protocol for Dephosphorylation of 5´-ends of DNA using rSAP in Restriction Enzyme Reaction (M0371)

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  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.