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Destruction of restriction site - (Mar/28/2007 )

Hello everybody,

i have a huge vector (~11kb) and I want to remove one restriction site. One is in the promoter, the other in the left border (at least I want to destroy the left border and insert it to a different site).
I made a partial digest with MunI and I elute the linear Plasmid from a 0,8% agarose gel using Qiaquick gel extraction kit.
Then I did MBN (NEB) treatment to remove the 5' ends. Re-ligate the vector (T4 DNA Ligase, MBI) and transformed it into INF alpha cells. --> I didn't receive colonies!
I also tried large Klenow --> the same, no colonies.
The competent cells were tested, and are ok.

Any ideas?

Thanks for help! ph34r.gif


11 kb is quite large to do site directed mutagenesis, but I would clone the sequence surrounding the site you want to remove into a cloning vector (pUC, pBKS or any other), perform site directed mutagenesis and clone it back into the 11 kb vector.