Protocol for Dephosphorylation of 5′ ends of DNA using Quick CIP (NEB #M0525)
- 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**
- Incubate at 37°C for 10 minutes.
- 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