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  • A-Tailing with Taq Polymerase

    Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 

    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.