I have been having trouble with what should be a fairly simple ligation. I had a plasmid that I needed to cut part out of, so I designed a couple PCR primers to do that. The plasmid already had a BamHI site on 1 side of the region I wanted to remove, so I made the other PCR primer with a BamHI site on its 5' end with 3 extra Cs so the restriction enzyme would bind better. Here is a crude diagram of what I tried to do:
So I did the PCR and BamHI digest, it looked good on gel, so I purified it from the gel, got the concentration on the Nanodrop, and set up a ligation.
However, I keep getting zero colonies. I have tried a 24 hour reaction at 4 degrees, and 10 minute reaction at room temp. I am currently trying a reaction where I supplement the buffer with fresh ATP, because the buffers are a little old. If that works, problem solved, if not, I am asking how to troubleshoot this.
This seems like it should be relatively simple, because I have no insert, just trying to close an empty vector, which seems to happen on its own when I don't want it to and dephosphorylate the vector.
The reaction with extra ATP did not produce colonies. I will keep trying. One idea I had was to do the PCR and then try blunt end ligation, since the Pfu polymerase should make blunt ends. Also got a new sample of ligase.