ligation problem - (Oct/31/2007 )

Hi all,
I have digested my vector with Nco I and Bst E II. the size of the fragments are 11 kb and 2 kb. I also digsted the second vector in which i am intresting a 600 bp as a isert with similar enzyme, I ligated the gel purified insert (600 bp) with non purified vector. and i got positive clone which was confirmed by PCR and R.E. But the size of the required construct is smaller then expected (9 kb compare to 11 kb). I have used DH5 alpha e,coli for my clone preperation. I do not unserstand what happen to my clone? why the deletion of some fragment was observed.

I have also try the ligation of vector and insert after CIP treatment and purification but on that cas i coud not get a single colony.
i generally use 1:3, 1:6 and 1:10 vector :insert ratio.

i stuck with this clone from atleast 6 months

Any suggestion will be welcome

Preeti Desai
Ph.D student

-preetindesai-

Did u check whether these restriction sites are in ur insert?????

-newarray-

Is your vector prone to recombine? Then this could lead to the loss of the small fragments. You would need to transform in bacteria which prevent recombination events like SURE cells.

-scolix-

QUOTE (newarray @ Oct 31 2007, 11:26 PM)

Did u check whether these restriction sites are in ur insert?????

Thanks for reply

yes i have chacked presence of R.E sites in both vector.Single site is found for both R.E in vector while in inser containing vector two site for NCO1 and 3 sites for BstE II are found. And i used gel purified insert for ligation.

Preeti

-preetindesai-