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