Feb 14, 2022
Version 4
Dephosphorylation of 5´-ends of DNA using rSAP in Restriction Enzyme Reaction (M0371) V.4
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
External link: https://www.neb.com/protocols/2013/06/10/protocol-for-dephosphorylation-of-5-ends-of-dna-in-restriction-enzyme-reaction-m0371
Protocol Citation: New England Biolabs 2022. Dephosphorylation of 5´-ends of DNA using rSAP in Restriction Enzyme Reaction (M0371). protocols.io https://dx.doi.org/10.17504/protocols.io.be59jg96Version created by New England Biolabs
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 15, 2020
Last Modified: February 14, 2022
Protocol Integer ID: 35745
Keywords: rSAP, Dephosphorylation,
Abstract
This protocol explains methods for dephosphorylation of 5´-ends of DNA using rSAP in restriction enzyme reaction (M0371).
Materials
MATERIALS
Shrimp Alkaline Phosphatase (rSAP) - 500 unitsNew England BiolabsCatalog #M0371S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Digest 1 µg –5 µg plasmid DNA 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
Scale larger reaction volumes proportionally.
Incubate at 37 °C for 01:00:00 or follow manufacturer’s recommendations.
Add 1 unit of rSAP for every 1 pmol of DNA ends (about 1 µg of a 3 kb plasmid).
Incubate at 37 °C for 30-60 minutes.
Stop reaction by heat-inactivation of rSAP and restriction enzyme (follow manufacturer's recommendations).
Note
If restriction enzyme cannot be heat-inactivated, DNA purification is required before ligation.