random plasmid mutagenesis

Michael Benedik bchs1b at Jane.UH.EDU
Thu Aug 12 22:47:13 EST 1993


In article <drose.745170941 at husc.harvard.edu>, drose at husc4.harvard.edu (David Rose) writes:
>
>Hello:
>
>	I am going to be doing a plasmid shuffle screen for mutants, and
>am trying to decide what kind of mutagenesis to do.  The gene in question
>has already been mutagenized with hydroxylamine, and I am interested in
>other methods that make other basepair changes.  I had considered doing
>PCR mutagenesis, but my gene has lousy sites and subcloning after
>amplification would be a pain.  So, I would ideally like a method, like
>hydroxylamine, in which one simply zaps the intact plasmid.  Is anyone
>using any other methods, like different chemicals, passage through a
>mutator bacteria strain, etc?  What works, what doesn't?  Any info would
>be appreciated.
>
>						Dave Rose
>						drose at husc.harvard.edu
>

You can mutagenize plasmid DNA in vitro with nitrous acid just like
with hydroxylamine. PAssing through a mutator strain like mutD works
fine, and many people treat E.coli carrying the plasmid with mutagenes
and then miniprep the DNA and transform for screening. Basically
almost everything works, just be sure to do some controls so that you
know what really is happening.


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 Michael Benedik				INTERNET: Benedik at uh.edu
 Dept. of Biochemical & Biophysical Sciences	
 University of Houston				BITNET: Benedik at uhou
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