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

    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.