Dephosphorylation of 5´-ends of DNA using rSAP in Restriction Enzyme Reaction (M0371)
New England Biolabs
Published: 2022-02-14 DOI: 10.17504/protocols.io.be59jg96
Abstract
This protocol explains methods for dephosphorylation of 5´-ends of DNA using rSAP in restriction enzyme reaction (M0371).
Before start
Steps
1.
Scale larger reaction volumes proportionally.
Digest 1µg–5µg in a 20 μl reaction as follows:
| A | B |
|---|---|
| DNA | ≥ 1 μl |
| Restriction Enzyme Buffer (10X) | 2 μl |
| Restriction Endonuclease | 1 μl |
| H2O, purified | to 20 μl** |
Note
2.
Incubate at 37°C for 1h 0m 0s or follow manufacturer’s recommendations.
3.
Add 1 unit of rSAP for every 1pmol of DNA ends (about 1µg of a 3 kb plasmid).
4.
Incubate at 37°C for 30-60 minutes.
5. If restriction enzyme cannot be heat-inactivated, DNA purification is required before ligation.
Stop reaction by heat-inactivation of rSAP and restriction enzyme (follow manufacturer's recommendations).
Note
