Home Protocols Protocol for Dephosphorylation of DNA 5´-ends using the Quick Dephosphorylation Kit (M0508)

Protocol for Dephosphorylation of DNA 5´-ends using the Quick Dephosphorylation Kit (M0508)

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  1. Prepare a 20 μl reaction as follows:

    DNA 1 pmol of DNA ends*
    CutSmart® Buffer (10X) 2 µl
    Quick CIP 1 µl
    H2O, purified to 20 µl**


  2. Incubate at 37°C for 10 minutes.

  3. Stop reaction by heat-inactivation at 80°C for 2 minutes.
*Note: 1 pmol of DNA ends is about 1 µg of a 3 kb plasmid.
**Scale larger reaction volumes proportionally.

Protocol for Dephosphorylation of DNA 5´-ends using the Quick Dephosphorylation Kit in a Restriction Enzyme Reaction (M0508)

  • The phosphatase can be added directly into the digestion reaction during or after DNA digestion
  • 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
  • Quick Cip is active in all NEB restriction enzyme buffers
  • The restriction enzyme should be heat-inactivated at the same time as the phosphatase after digest and dephosphorylation
  • If the restriction enzyme(s) cannot be heat-inactivated, DNA purification is required before ligation