Feb 14, 2022

Public workspaceNon-radioactive phosphorylation with T4 PNK (M0201) V.2

DOI
dx.doi.org/10.17504/protocols.io.bdd2i28e
  • 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.bdd2i28e
External link: https://www.neb.com/protocols/0001/01/01/non-radioactive-phosphorylation-with-t4-pnk-or-pnk3-phosphatase-minus
Protocol Citation: New England Biolabs 2022. Non-radioactive phosphorylation with T4 PNK (M0201). protocols.io https://dx.doi.org/10.17504/protocols.io.bdd2i28eVersion 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 08, 2020
Last Modified: February 14, 2022
Protocol Integer ID: 33946
Keywords: phosphorylating, T4PNK reaction buffer, PNK, T4 PNK 3' phosphatase, M0201
Abstract
Protocol for non-radioactive phosphorylation with T4 PNK.
Materials
MATERIALS
ReagentAdenosine-5 Triphosphate (ATP) - 1 mlNew England BiolabsCatalog #P0756S
ReagentT4 Polynucleotide Kinase - 500 unitsNew England BiolabsCatalog #M0201S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Set up the following reaction in a microcentrifuge tube TemperatureOn ice :
AB
ReagentVolume
DNAup to 300 pmol of 5´ termini
T4 PNK Reaction Buffer (10X)5 µl
ATP (10 mM)5 µl
T4 PNK1 µl (10 units)
Nuclease-free Waterup to 50 µl

Note
  • ATP is not supplied
  • 1X T4 DNA Ligase Buffer contains 1 mM ATP and can be substituted in non-radioactive phosphorylations (T4 Polynucleotide Kinase exhibits 100% activity in this buffer).

Pipetting
Incubate at Temperature37 °C for Duration00:30:00 .
Incubation
Heat inactivate by incubating at Temperature65 °C for Duration00:20:00 .
Incubation