Protocol for Dephosphorylation of DNA 5´-ends using the Quick CIP in a Restriction Enzyme Reaction (NEB #M0525) 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 µl of Quick CIP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and incubate at 37°C for 10 minutes.

  4. Stop reaction by heat-inactivation of Quick CIP and restriction enzyme (follow manufacturer's recommendations).
Note: The vector digest and dephosphorylation with Quick CIP can be performed in one reaction at the same time. If restriction enzyme cannot be heat-inactivated, DNA purification is required before ligation.