I have done lot of ligations, transformations in my previous lab, but I have never encountered such problem, which was asked by a junior at my current place. The cloning that he is doing is very simple-
he has 1kb insert that he cloned in pGEMT vector first, using the kit. he got the clones, and then he cut the insert using the sites BamH1 and Hind III. Now he wants to clone it in pET28a vector to express his protein. He is using JM109 competent cells. Whenever he transforms his ligated mix, he never gets colonies. I told him to try all sorts of things that we generally tell...to check the comp cells efficiency, ligase etc. His comp cells are fine as he always gets colonies with pET plasmid. Ligase also seems to be working as he loaded both ligated product and empty plasmid on gel and he observed a shift in ligated product. I am attaching the gel picture herewith. I don't know what is going wrong. Molecular biology experts, please help to solve this problem.