Feb 21, 2022

Public workspaceDephosphorylation of 5´-ends of DNA using rSAP (M0371) V.2

DOI
dx.doi.org/10.17504/protocols.io.bg9kjz4w
  • 1New England Biolabs
  • New England Biolabs (NEB)
    Tech. support phone: +1(800)632-7799 email: info@neb.com
New England Biolabs
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.bg9kjz4w
External link: https://www.neb.com/protocols/2013/06/10/protocol-for-dephosphorylation-of-5-ends-of-dna-m0371
Protocol Citation: New England Biolabs 2022. Dephosphorylation of 5´-ends of DNA using rSAP (M0371). protocols.io https://dx.doi.org/10.17504/protocols.io.bg9kjz4wVersion 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: June 07, 2020
Last Modified: February 21, 2022
Protocol Integer ID: 37900
Keywords: dephosphorylating 5' ends of DNA, Dephosphorylation using rSAP, phosphatase, dephosphorylate
Abstract
Protocol for Dephosphorylation of 5´-ends of DNA using rSAP (M0371).
Guidelines
Dephosphorylation of 5´- ends of DNA in Restriction Enzyme Reaction
  • The phosphate can be added directly into the digestion reaction during or after DNA digestion
  • rSAP is active in all NEB restriction enzyme buffers
  • The restriction enzyme should be heat inactivated at the same time as the phosphatase after digest and dephosphorylation
  • If restriction enzyme(s) cannot be heat inactivated, DNA purification is required before ligation
Materials
MATERIALS
ReagentShrimp Alkaline Phosphatase (rSAP) - 500 unitsNew England BiolabsCatalog #M0371S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Prepare a Amount20 µL reaction as follows:
AB
DNA1 pmol of DNA ends*
CutSmart® Buffer (10X)2 µl
rSAP1 unit
H2O, purifiedto 20 µl**
Note
* 1 pmol of DNA ends is about 1 µg of a 3 kb plasmid
Note
**Scale larger reaction volumes proportionally

Pipetting
Incubate at Temperature37 °C for Duration00:30:00 .
Incubation
Stop reaction by heat-inactivation at Temperature65 °C for Duration00:05:00 .