Protocol for Dephosphorylation of 5´-ends of DNA using CIP (NEB #M0290)
Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community.
- Prepare a 20 μl reaction as follows:
DNA 1 pmol of DNA ends* CutSmart® Buffer (10X) 2 μl CIP 1 unit H2O, purified to 20 μl**
- Incubate at 37°C for 30 minutes.
- Purify DNA by gel purification, spin-column (NEB #T1020 or NEB #T1030), or
* Note: 1 pmol of DNA ends is about 1 μg of a 3 kb plasmid.
** Scale larger reaction volumes proportionally.
Dephosphorylation of 5´-ends of DNA in Restriction Enzyme Reaction
- The phosphatase can be added directly into the digestion reaction during or after DNA digestion
- CIP is active in all NEB restriction enzyme buffers
- DNA purification is required before ligation