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*
   rCutSmart™  Buffer (10X)	2 μl
   Quick CIP	1 μl
   Nuclease-free water	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