failure of insertion of fragment into large vector - (Oct/31/2005 )
I am doing a ligation between 2.4kb fragment and 10kb fragment.
The two fragment are cutted by SbfI and BamHI and ligate by 3:1(v/v). It is said that for large vector clone it is better using Stbl2 or XL1-Blue competent cells for transformation. So I used both.
But after transformation and restriction enzyme confirmation, there is no corret clone at all. The bands are confused. The bands on the gel are neither vector selfligated pattern nor insert selfligated pattern. I don't know what they are and why?
Does anyone have experience with doing large fragment cloning with Stbl2 or XL2?
Any suggestion will be welcome and thank you in advance.
I routinely clone large fragments my record is a 10.2 kb fragment into a 7.1kb vector, I generally use XL1-blue for my cloning.
Have you checked the concentrations of your fragments on a gel prior to ligation the yield difference will be very different between the 2 fragments, often large fragments result in quite low yields and you may need to use more of the larger fragment.
Also are you sure both enzymes are cutting both fragments?