In article <316d8p$ju1 at mserv1.dl.ac.uk>, Nelson Salazar <N.SALAZAR at lshtm.ac.uk> writes:
|>|> Hello there,
|> I am trying to clone two SacII DNA fragments (about
|> 6.5 and 7.5 kb in size) into pBluescript II separately, and I am not
|> getting recombinant plasmid after the transformation. I get many
|> colonies but when I screen them, they DO NOT have the insert DNAs
|> that I want, that is, the plasmid religates and gives me "false"
|>|> How could I improve the cloning of my inserts ?? and how could I
|> avoid religation of my vector? I think that in this case
|> dephosphorylating of the pBluescript SacII cut does not make any
|> sense ? Any suggestions will be greatly appretiated.
Did you ever try cutting after ligation with a restriction enzyme having a
site in the insert but not in the vector (if such a site exists)?
Sometimes you have to buy a weird enzyme, but this always sharply reduces
background for me.