DNA ligation

Michelle mlovingshimer at tamu.edu
Mon Mar 11 14:49:06 EST 2002


I am having extreme difficulty with a "simple" ligation and was
looking for some advice.  I am trying to ligate a 1 kB fragment
generated by PCR with restriction sites incorporated into the primers
(Hind III and EcoRI) into a Promega vector (pALTER-1).   I am getting
the PCR product just fine.  I have sequenced the ends of both the
uncut and cut PCR product to make sure the sites are intact and being
cut correctly and they are fine.  In order to avoid gel purifying the
digested insert and vector, I have been using Qiagen's Qiaquick PCR
cleanup kit.  I felt that this would be sufficient since the unwanted
pieces of DNA from the digests are quite small and presumably would
not stick to the column.  I have tried to use several different ratios
of vector:insert, but end up with no colonies.  I have not had great
transformation efficiency with my competent cell preps as I only get
20-30 colonies when transforming uncut vector.  I am in the process of
going back and doing some controls like re-ligating the vector after a
single cut to make sure my ligase is ok.  Any other suggestions??




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