A-Tailing with Taq Polymerase
New England Biolabs
Published: 2022-02-08 DOI: 10.17504/protocols.io.be6ajhae
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).
Before start
Steps
1. 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.
Clean-up the amplified DNA from the PCR components
Note
2.
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).
3.
Incubate the reaction at 72°C for 0h 20m 0s.
