polishing.... followed by ligation - I HAVE BEEN STRUCK FOR THE FIRST TIME ... (Jan/16/2005 )
Can any one could give suggetion.. My insert ( 1.8 kb) inside a cloning vector (3.2kb) was restricted with non palindromic Bstx1 at two sites in order to delete 700 bp resulting into 1.1 kb size. After the restriction the plasmid is in linearized form because of non palindromic sites.
I did polishing to creat blunt ends using T4 DNA polymerase from Fermentas .. and followed by ligation with rapid DNA ligation kit from Roche. The ligation mixture was transformed in to TOP 10 competent cells. But even repeating the things for more than 5 times I m anot getting the transformants.
I already tried with positive controls and my competent cells are giving quite a good transformant efficiency. I m not getting what is the problem.. either T4DNA polymerase or T4 DNA ligase is giving a problem. This time I m going to try PEG4000. In the mean while i'll wait for ur replies.
Could you try to liagte the insert into the vector at 22C overnight?