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  • Protocol for Dephosphorylation of 5´-ends of DNA using CIP in Restriction Enzyme Reaction (M0290)

    Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 

    Protocol

    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 CIP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and incubate at 37°C for 30–60 minutes.

    4. Purify DNA by gel purification, spin-column or phenol extraction.

    5. Proceed with ligation.