Protocol for Dephosphorylation of DNA 5´-ends using the Quick CIP in a Restriction Enzyme Reaction (NEB #M0525)
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- Digest 1–5 μg of plasmid DNA in a 20 μl reaction as follows:
DNA >1 µl Restriction Enzyme Buffer (10X) 2 µl Restriction Endonuclease 1 µl H2O, purified to 20 µl
Note: Scale larger reaction volumes proportionally.
- Incubate at 37°C for 60 minutes or follow manufacturer’s recommendations.
- 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.
- Stop reaction by heat-inactivation of Quick CIP and restriction enzyme (follow manufacturer's recommendations).