site-directed mutagenesis problems

Russell A. Maurer ram3 at po.CWRU.Edu
Wed Dec 9 21:24:06 EST 1992


In a previous article, wsun at jeeves.ucsd.edu (Fiberman) says:

>Hi, netters,
>
>I recently posted the problems I had and appreciate your
>responses.  However, I am still not getting the mutants using
>the Bio-rad kit.  I am getting transformants with the negative
>control DNA - where I make the second strand in the absense of
>any mutagenic primer. 
>
>Just recently, I found that my SS DNA was able to transform XL1
>blue competents.  Apparently the SS DNA  prep was contaminated
>with DS DNA.  Has anyone had experienced this problem?  I am
>making SS DNA from the Bluescript plasmid in CJ236 cells.  I am
>thinking of gel purifying the SS DNA.  Is this a logical or a
>stupid idea?  I appreciate any comments.
>
>-fm
>
>I doubt purification of ssDNA is what you need, as ssDNA transforms
E. coli quite well provided it has some way to prime synthesis of
the complementary strand (as Bluescript certainly does).

Russ Maurer



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