Protocol for Dephosphorylation of 5´-ends of DNA using CIP (NEB #M0290) 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*
    CutSmart®  Buffer (10X) 2 μl
    CIP 1 unit
    H2O, purified to 20 μl**

  2. Incubate at 37°C for 30 minutes.

  3. Purify DNA by gel purification, spin-column (NEB #T1020 or NEB #T1030), or
    phenol extraction.

* 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
  • CIP is active in all NEB restriction enzyme buffers
  • DNA purification is required before ligation