ligation problem - (Oct/31/2007 )
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
Did u check whether these restriction sites are in ur insert?????
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.
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.