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  • Protocol for Dephosphorylation of 5´-ends of DNA using CIP (NEB #M0290)

    Protocols.io 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 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 phosphate 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