Protocol Online logo  Selection of clones after ligation? -
(Oct/07/2011 )  Pages: Previous 1 2   If you are just EtOH
ppting your cut oligo, there is a good chance that the ends of
the ds oligo are interfering with your ligation. Each end will
have a compatible overhang with your vector on one side, and a
blunt end on the other. These little ds oligo "nubs" can just
act as a cap to your vector, preventing proper ligation.
However, since you are cutting your oligo, then all fragments
(the overhangs of both ends and both sides of the insert) will
have 5' Phosphates.  I do understand that you cut your vector
with 2 different enyzmes, and so you think you don't need to
phosphatase treat your cut vector. This is a mistake that a lot
of people make. There is a difference between what theory tells
you is supposed to happen versus what reality tells you actually
does happen. Unless you perform a vector only ligation and
transformation and get ZERO colonies on a selection plate, then
there is always the risk of vecto