Will insert increase empty vecotr recircularization? - (Aug/09/2008 )
Hi, I'm trying to ligate isolated HSV-TK into a SalI linearized vector. Following electroporation substantially more colonies were present on the vector+insert plates than the vector only control plates. However, after extensive Southern blot hybridisation screening non of the colonies were positive for HSV-TK. Could the presence of the insert be increasing vector self ligation?
Thanks for your help!
In that case i'd be worried about your hybridisation. How about a standard miniprep and digest to screen your clones? That will show you whether the clones are positive or not (which they seem to be). I don't think the insert can increase vector self ligation. Not sure how that would work.
I like crack gels for screening colonies like that: pick a bunch of colonies, dip them into a crack buffer, run agarose gel.
My rule of thumb is that the vector plus insert must have more than 2x as many colonies as vector only plus ligase. If it's less than two-fold, most of the colonies will be vector only.
Thanks for the advice, i like the sound of the crack gel method, will give that a go.