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!

-lce-

QUOTE (lce @ Aug 10 2008, 05:27 AM)

substantially more colonies were present on the vector+insert plates than the vector only control plates

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.

-killerkoz17-

I like crack gels for screening colonies like that: pick a bunch of colonies, dip them into a crack buffer, run agarose gel.

-Andriy-

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.

-tfitzwater-

Thanks for the advice, i like the sound of the crack gel method, will give that a go.

-lce-