Apr 12, 2022
Version 2
Non-radioactive phosphorylation with T4 PNK 3' phosphatase minus (M0236) V.2
- 1New England Biolabs
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 3' phosphatase minus (M0236). protocols.io https://dx.doi.org/10.17504/protocols.io.4rm7vzdrlx1w/v2Version 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: May 25, 2020
Last Modified: April 12, 2022
Protocol Integer ID: 37461
Keywords: phosphorylating, T4PNK reaction buffer, PNK, T4 PNK 3' phosphatase
Abstract
Protocol for non-radioactive phosphorylation with T4 PNK 3' phosphatase minus.
Materials
MATERIALS
Adenosine-5 Triphosphate (ATP) - 1 mlNew England BiolabsCatalog #P0756S
T4 Polynucleotide Kinase (3' phosphatase minus) - 200 unitsNew England BiolabsCatalog #M0236S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Set up the following reaction in a microcentrifuge tube On ice :
| A | B | |
| Reagent | Volume | |
| DNA | up to 300 pmol of 5´ termini | |
| T4 PNK Reaction Buffer (10X) | 5 µl | |
| ATP (10 mM) | 5 µl | |
| T4 PNK | 1 µl (10 units) | |
| Nuclease-free Water | up 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).
Incubate at 37 °C for 00:30:00 .
Heat inactivate by incubating at 65 °C for 00:20:00 .