Can't find a recombinant after ligating - (Apr/09/2013 )
I'm trying to ligate a 1kb insert into a 5kb destination vector. Unfortunately, I am only able to use a single restriction site (Sal1); therefore, the cut plasmid can easily religate. I ligated insert and vector at a 3:1 ratio. I had to have screened over 60 colonies so far and not one was a recombinant (PCR and restriction digest analysis).
EDIT: And I am certain that my insert is being cut since I first put it into a topo construct.
Did you de-phosphorylate the vector?
No, we have CIP, but for whatever reason my PI advised against it.
Unfortunately he is a bit correct, CIP can, and often does, cause a lot of problems, but as you are using a single cut, it should at least help to prevent the plasmid re-ligating, which would tell you if there is a problem with the insert or the ligation.
I'm giving it a go, we'll see how it turns out.