random plasmid mutagenesis

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Fri Aug 13 01:29:00 EST 1993


--------------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
********************

Hi Dave!
I have used the E.coli mutator mutD5 with success!  I got two strains 
containing the mutation from Craig Giroux (Wayne State University).  The way
it works is that the mutator is conditional on the growth media.  If you 
grow on M9 medium the mutator effect is minimized so you do all the 
manipulation
on this medium, like putting in the plasmid, etc.  When you want the mutator
to kick in just grow in on LB and you get 30K - 40,000 times over wildtype 
levels
of mutation.  Under inducing conditions 85% are transitions, 10% are 
transversions
and 5% are single base frameshifts.  Papers looking at this mutator are
R.G. Fowler et al MGG 133, 179-191(1974) an oldie
R.M. Schaaper, PNAS 85 8126-8130 (1988)
If I remember correctly Pat Foster (BU) works with this gene.  She has posted
many times to the methods board so you can probably get her E mail add from 
the user list. 
Good Luck 
Dan Gietz
______________________________________
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)789-3458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
"Trying to do the Manitoba Thing"
_______________________________________



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