ligation only concatenate and not circularize - (Jun/23/2006 )
I've a bit problem since some weeks : i do classic ligations and E. coli transformation and never obtain any clone.
In that way i've try to control my ligations and i can observe strange results : when i ligate empty plasmid with the two same restriction sites and i control it on agarose gel i only see concatenation and not any trace of circular plasmid (on gel i only see bands with bigger molecular weight compared with the linearised plasmid).
I'm not really expert in that kind of technics so i would be happy if someone know what kind of parameters could help me to obtain circular plasmid instead of concatenations (i generally use classic conditions following the vendors instructions, like using approximatelly 100 ng of plasmid for a 20 µl ligation and ligating for 3 hours at 21 °C)
Sorry for my poor english writting, i'm not fluent.
Thanks for advance.
The statistics for recircularization are controlled by the DNA concentration. Dilute reactions favor circularization, and the optimum is lower than the concentration you are using. Once the concatenation starts, the longer DNA fragments recircularize less frequently, so there is a nonlinear term.
Thanks for your answer,
I will try with lower concentrations and see if i can get better results.