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religation of vector DNA

Frank Anthony Skraly skralyf at cae.wisc.edu
Thu Jul 28 17:03:54 EST 1994


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" 
|> transformants.
|>  
|> 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.

F.S.



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