Feb 07, 2022
Version 2
A-Tailing with Taq Polymerase V.2
- 1New England Biolabs
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs 2022. A-Tailing with Taq Polymerase. protocols.io https://dx.doi.org/10.17504/protocols.io.be6ajhaeVersion 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 07, 2022
Protocol Integer ID: 35746
Keywords: A-tail, Taq, Taq pol, Taq polymerase
Abstract
This protocol can be used to add As to the blunt-ends of DNA fragments that have been amplified using a high-fidelity polymerase (such as Q5® High Fidelity DNA Polymerase).
Materials
MATERIALS
ThermoPol Reaction Buffer Pack - 6.0 mlNew England BiolabsCatalog #B9004S
Taq DNA Polymerase with ThermoPol Buffer - 400 unitsNew England BiolabsCatalog #M0267S
Safety warnings
Please refer to the Safety Data Sheets (SDS) for health and environmental hazards.
Clean-up the amplified DNA from the PCR components
Note
This can be done by using a PCR-column purification protocol. This step is essential because the robust exonuclease activity associated with the high-fidelity enzyme will remove any untemplated nucleotides that are added by Taq DNA Polymerase.
Set-up the reaction by adding the following components:
| A | B | |
| REAGENT | AMOUNT | |
| PCR-amplified DNA | X µl | |
| 10X ThermoPol® Buffer (NEB#B9004) | 5 µl | |
| 1mM dATP | 10 µl | |
| Taq DNA Polymerase (NEB#M0267) | 0.2 µl | |
| H2O | X µl | |
| Total Reaction Volume | 50 µl* |
*This volume can be adjusted based on the volume of PCR-amplified DNA that needs to be added).
Incubate the reaction at 72 °C for 00:20:00 .