Feb 14, 2022

Public workspaceDephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289) V.3

DOI
dx.doi.org/10.17504/protocols.io.bd2xi8fn
  • 1New England Biolabs
  • New England Biolabs (NEB)
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DOI: dx.doi.org/10.17504/protocols.io.bd2xi8fn
External link: https://www.neb.com/protocols/0001/01/01/vector-dephosphorylation-protocol
Protocol Citation: New England Biolabs 2022. Dephosphorylation of 5´-ends of DNA using Antarctic Phosphatase (NEB #M0289). protocols.io https://dx.doi.org/10.17504/protocols.io.bd2xi8fnVersion 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: March 21, 2020
Last Modified: February 14, 2022
Protocol Integer ID: 34615
Keywords: Dephosphorylation, 5’ ends, Antarctic , Phosphatase, M0289,
Abstract
This is the protocol for dephosphorylation of 5'-ends of DNA using AnP (Antarctic Phosphatase - M0289).
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
  • Antarctic Phosphatase is active in all NEB restriction enzyme buffers only when supplemented with Antarctic Phosphatase Reaction Buffer, which provides Zn2+ required for enzyme activity
  • The restriction enzyme should be heat inactivated at the same time as the phosphatase after digest and dephosphorylation
  • If restriction enzyme cannot be heat inactivated, DNA purification is required before ligation
Materials
MATERIALS
ReagentAntarctic Phosphatase - 1,000 unitsNew England BiolabsCatalog #M0289S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Prepare a Amount20 µL reaction as follows:
AB
COMPONENTAMOUNT
DNA1 pmol of DNA ends*
Antarctic Phosphatase Reaction Buffer (10X)2 μl
Antarctic Phosphatase5 units
H2O, purifiedto 20 μl**
* Note: 1 pmol of DNA ends is about 1 μg of a 3 kb plasmid.
** Scale larger reaction volumes proportionally.

Pipetting
Incubate at Temperature37 °C for Duration00:30:00 .
Incubation
Stop reaction by heat-inactivation at Temperature80 °C for Duration00:02:00 .