Random plasmid mutagenesis

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Fri Oct 15 14:11:00 EST 1993


Hi Jari!
    I have used the method of Rose and Fink 1987 Cell 48:pp1047
   Dissolve 10ug of precipitated plasmid DNA in
    500 ul of hydroxylamine mix (0.09 gm of NaOH, 0.35gms of hydroxylamine HCl
    in 5 mls of ice cold water made fresh before use)
    Incubate at 37^o C for 20 hrs
    terminate by adding 10ul of 5 M NaCl and 50 ul of 1mg/ml of BSA
    and 1 mls of 95% ethanol.
    incubate at -70 for some time and then spin to pellet DNA
    Dissolve DNA in TE and use to transform E.coli or yeast or what ever.

You can also used passage through a mutator E.coli strain like mutD5
here is a copy of a message I post earlier about this!

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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From: jvehmaa at convex.csc.fi (Jari Vehmaanper)
Subject: Random plasmid mutagenesis
Date: 15 Oct 93 08:58:09 GMT
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Hallo netters,

 I would like to hear your experiences in chemical mutagenesis of
*double* stranded plasmid DNA for introducing random single or double
base substitutions. I have a regular pBluescript- and pSC101-derived
plasmids of about 4 kb in size, and screening is done in E.coli. I want
to keep it simple, so I prefer chemicals to any oligo-based mutagenesis
and I also want to avoid isolating single-stranded DNA (but I try to keep
my mind open).

I have tried a couple times with hydroxylamine, but with
hydroxylamine-O-sulphonic acid I lost much of my DNA after dialysis and
EtOH-precipitation, and with hydroxylamine-HCl (which I understand is
same as hydroxylammonium chloride) I did not get mutants, as judged by no
loss of transformation frequency and no more white colonies after
treatment. I treated my DNA with HA upto 2 h at 75 C.

Any suggestions are welcome. Thank you in advance for taking the trouble.

Jari Vehmaanpera
E-mail jvehmaa at convex.csc.fi




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