HomeFAQsI'd like to clone a fragment amplified with Phusion® Hot Start Flex 2X Master Mix. Do I have to blunt-end clone?
FAQ: I'd like to clone a fragment amplified with Phusion® Hot Start Flex 2X Master Mix. 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 all the Phusion Hot Start Flex 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 ) 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.