random mutagenesis

CHAD A. RAPPLEYE rappleye at biomail.ucsd.edu
Tue Nov 21 16:07:07 EST 2000


Chemical mutagenesis is quite feasible.  For example, just treating the
BAC-harboring E.coli with any of the standard chemical mutagens (EMS,
hydroxylamine, nitrosoguanidine) will introduce random mutations.  Look in
J.H. Miller's book "Experiments in Molecular Genetics" for the appropriate
protocols.  Mutagenesis is easy and effective: suspend the culture
in the mutagen for a few hours, wash, and plate for your screen.  With
E.coli mutator
strains, I imagine the rate of mutagenesis is lower than with the chemical
mutagens which will mean screening through a lot more.

Chad Rappleye

Aroian Lab
UC, San Diego
(858) 822-1397

http://www.biology.ucsd.edu/labs/aroian

On Tue, 21 Nov 2000, Daniel Schumacher wrote:

> Hello,
> I hope somebody can help me. I=B4m looking for a method /protocol to intr=
oduce
> mutations randomly in a BAC-Vector.
> I already looked for chemicals that I could just add to the media, which =
I
> thought would be the easiest way, but wasnt lucky in finding some method.
> Also I thought of uv-light ? By the way the bacteria i use are E.coli.
> Most of the methods I found were using random PCR, which is OK if you onl=
y
> have some kb but not with 180 kb.
> So if anybody could give me a hint of what chemical to use and how I woul=
d
> be very happy!
> I also heard about mutator-strains which can be used for this kind of thi=
ng.
> Does anybody know about them?#
>=20
> THX
>=20
> Daniel
>=20
>=20
>=20
>=20






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