Protocol for Dephosphorylation of 5′ ends of DNA using Quick CIP (NEB #M0525)

  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.

Dephosphorylation of DNA 5′-ends using Quick CIP in a Restriction Enzyme Reaction

  • 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 restriction enzyme(s) cannot be heat inactivated, DNA purification is required before ligation

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Quick CIP