FAQ: I'd like to clone a fragment amplified with Phusion® High-Fidelity DNA Polymerase. Do I have to blunt-end clone?

Blunt-end cloning is recommended. However, if TA cloning is required, 3´A-overhangs can be added with a different polymerase. It is very important to remove the Phusion® High-Fidelity DNA Polymerase first by purifying the PCR product. The proofreading activity of Phusion is very strong, so any residual polymerase will degrade the A overhangs as they are added. Taq DNA Polymerase (NEB# M0267, NEB# M0273) or Klenow(exo-) DNA Polymerase (NEB# M0212) are excellent options for A-overhang addition. We recommend ligating immediately so the 3´A-overhangs will not be lost during storage.

Phusion® is a registered trademark of Thermo Fisher Scientific.