Pharmacia U.S.E. Mutagenesis

wetsel_r at wums.wustl.edu wetsel_r at wums.wustl.edu
Sun Jan 23 11:50:00 EST 1994


In a previous article, sschendel at bilbo.bio.purdue.edu (Sharon Schendel) wrote:
>Just a short note concerning U.S.E. Mutagenesis, in particular, 
>Pharmacia's kit.  I had been trying to make mutants using 
>Stratagene's pSKBluescript for my vector.  The kit's manual 
>makes the claim that plasmids which have lost the unique 
>restriction site have a 90% chance of gaining the desired 
>mutation.  I found three such plasmids, but upon sequencing, 
>found that they had only lost the selection site... they had 
>not gained my mutation.  Pharmacia's technical service rep 
>informed me that for some "unknown reason" pSKBluescript will 
>not work with their kit.  Those plasmids which they could 
>guarantee would work are pBR322 and the pUC vectors (i.e. those 
>marketed by Pharmacia).  Just thought that I'd share this, 
>since there is no mention of plasmids that would not work 
>with the U.S.E. method either in Pharmacia's manual or in the 
>original Deng and Nickoloff paper (Anal. Biochem 200,81).
> 
>Sharon Schendel
>sschendel at bilbo.bio.purdue.edu

This is bizzare Sharon...  From what I could tell, I was under the 
assumption that pBluescript was DERIVED from pUC19.  So in theory, it 
should work.  I know someone out there will ask for my source but I 
distinctly remember reading that the backbone of pBS was initialy derived 
from pUC19 and then S_Gene added additional restriction sites, and the f1, 
but the ori, B-gal, and amp all came from puc.


Anyone correct if I'm mistaken but I'll also check again myself.
David
haviland at kids.wustl.edu
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