Dephosphorylation of 5´-ends of DNA using CIP (M0290)
New England Biolabs
Published: 2022-02-22 DOI: 10.17504/protocols.io.bddbi22n
Abstract
Protocol for Dephosphorylation of 5´-ends of DNA using CIP in Restriction Enzyme Reaction. Uses the Calf Intestinal Alkaline Phosphatase (CIP - M0290).
Before start
Steps
1.
Prepare a 20µL reaction as follows:
| A | B |
|---|---|
| COMPONENT | AMOUNT |
| DNA | 1 pmol of DNA ends* |
| CutSmart® Buffer (10X) | 2 μl |
| CIP | 1 unit |
| H2O, purified | to 20 μl** |
- Note: 1 pmol of DNA ends is about 1 μg of a 3 kb plasmid.** Scale larger reaction volumes proportionally.
2.
Incubate at 37°C for 0h 30m 0s.
3.
Purify DNA by gel purification, spin-column (NEB #T1020 or NEB #T1030) or phenol extraction.
