restriction enzyme pcr cloning
Posted 17 April 2018 - 08:13 AM
Im trying to do pcr cloning of a gene by adding 2 different restriction enzymes to the pcr primers.
The enzymes I plan to use are EcorV and not1 at pcdna3.0 multiple cloning site. Ecorv yields blunt ends while not1 yields sticky ends. Is this possible? Both enzymes are compatible with a single buffer from NEB.
Im new to cloning and would appreciate any advice.
Posted 17 April 2018 - 12:54 PM
It should be fine. Note that blunt end cloning is orders of magnitude less efficient than sticky end. Make sure that you have a few bases (3-6 usually) 5' of the restriction site on the primers, so that the restriction enzymes have something to hold onto when cutting.