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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).
1. Clean-up the amplified DNA from the PCR components. 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.
2. Set-up the reaction by adding the following components:
- PCR-amplified DNA – X
- 10X ThermoPol® Buffer (NEB #B9004 )– 5ul
- 1 mM dATP – 10 µl
- Taq DNA Polymerase (NEB #M0267 ) –0.2 ul
- H2O – X
- Total Reaction Volume – 50 ul (This volume can be adjusted based on the volume of PCR-amplified DNA that needs to be added).
3. Incubate the reaction at 72 0C for 20 minutes.