Protocol for Dephosphorylation of 5´-ends of DNA using Quick Dephosphorlyation Kit (M0508)
- 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**
- Incubate at 37°C for 10 minutes.
- Stop reaction by heat-inactivation at 80°C for 2 minutes.
** Scale larger reaction volumes proportionally.
Dephosphorylation of DNA 5´-ends using the Quick Dephosphorylation Kit 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