High transformation background - (May/14/2007 )
I am struggling with transforming E. coli to carry a ligation product. After O/N ligation at RT (not confirmed on a gel after ligating), I add 1uL ligation mix to 10uL purchased competent cells. I incubate 30'-1h on ice, heat shock at 42C 1' the rescue in 100-125uL SOC media. I plate the rxn on fresh (1-2 week old) LB-amp plates (0.1g amp added at 55C to 1L LB). After O/N incubation at 37C, the plate with vector alone (no insert) has a full lawn of colonies, perhaps less than the vector + insert, but ALOT of colonies. Any suggestions for reducing the colonies on this plate???
I have one question though, did you dephosphorylate your vector? A lawn of colonies, especially one with vector alone is not right. Is the vector fully cut? A vector only plate should either be clean or have at most 10s of colonies.
Dephosphorylation of vector is the first question, yes. But also have you tried plating just your compentent cells on your plate? I once had the same problem and it turned out due to the amp was not good.
Another question is: ligate o/n at RT? I only did 15min-1h at RT with the Rapid Ligation (Roche). If I do o/n, it would be at 16-18oC or so. Is it right?
Check the composition of your ligase buffer - if it contains polyethylene glycol it means you have a "rapid ligation" buffer and should only ligate for 15 minutes or so. The ligation efficiency decreases with time, for some reason (according to Invitrogen). But that wouldn't explain your results. I think you have a phosphorylated vector or your vector is not completely cut (as perneseblue suggested).