Self Circularization Problem with a 6KB vector - self ligation of a 6KB vector (Oct/30/2007 )
I have a question about how to make a vector religate to itself instead of forming concatamers.
The 6KB vector was digested with BamHI, cleaned up by PCR purification kit (Qiagen), and later on put in a ligation reaction at the concentration of 5ng/ul for 3 hours at room temperature.
The ligation reaction was set up as follows:
vector 2 ul
ligase buffer(10x) 1ul
T4 ligase(Invitrogen) 1ul
add water to 10ul total
After transformation, there were no colonies.
I assumed that at least some of the fragments will recircularize even though the condition favors the formation of concatamers.
But it seems that my ligation reactions didn't work after adjusting the vector concentration from 10ng/ul to 5ng/ul.
Does anyone have similar kind of experience or any suggestions?
How was your positive control? Without it, you don't know whether your compotent cells are good, your transformation procedures are OK.
After ligation, run a gel using the ligation reaction along side with uncut plasmid, negative ligation, it will tell you clearly whether you get concatamers or circularized plasmid.