XL1-Red in vivo mutagenesis

Michael Benedik benedik at uh.edu
Mon Apr 29 09:43:12 EST 1996


In article <4lolvs$1hu at mserv1.dl.ac.uk>
neuro <meisel at neuro.charite.hu-berlin.de> writes:

> We are trying random mutagenesis of a 9kb plasmid using the XL1-Red in vivo 
> mutagenesis system (Stratagene). Even if we follow the instructions of the 
> manual, we can't find any mutations in the gene of interest. The gene, a 
> restriction/modification system, is not essential for bacterial survival, 
> except for a mutation effecting the modification function . The mutation 
> frequency of the XL-1 Red  strain should be 1 per 2000 bp per 30 
> generations, as ruled out by the suppliers. We sequenced more than 10kb 
> without any mutation after 40-50 generations. The technical service of 
> Stratagene has no explanation for this. Is there anybody having the same 
> experience or some better. Or is there anybody having an idea for efficient 
> random in vivo mutagenesis of a plasmid.
> 
> Andreas Meisel	
> Exp. Neurology
> Humboldt University Berlin
> meisel at neuro.charite.hu-berlin.de
> 

We have done similar things with good success, but using standard mutD
strains and not XL1 red. The first thing I would check is whether you
are getting good frequency of mutations. Take a plasmid with an easy to
screen marker (like pUC18 or pBluescript) and propagate in XL1-Red for
same number of generations, then transform into your favorite
blue/white strain and determine the ratio of white colonies to blues.
Doing appropriate controls (standard non mutageneic strain or XL1 red
under nonmutagenic conditions) should demonstrate if your procedure is
in fact giving you mutations.




Michael Benedik
Department of Biochemical Sciences
University of Houston
benedik at uh.edu



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