classical mutagenesis? the best way to do it
rafael at howard.genetics.utah.edu
Mon Feb 5 19:45:32 EST 1996
On 5 Feb 1996, Christian Belanger wrote:
> I'm looking for a regular protocol to do a non specific mutagenesis on
> E.coli. Which method is the better? Which chemical will give the best
Are you talking of chemical mutagenesis? I love diethyl sulfate method
(DES). It is easy to use, not excesivelly volatile and because is a
liquid, you don't need to weighth any mutagenic dust. Basically, add 1
drop (50 ul) of DES to 1 ml of cells; incubate at 37 during 5-30 minutes
(you will have to determine the best mutants/death ratio; normally in
enterobacteria 1% of supervivency is perfect; that is got using 20
minutes. Don't forget use a recA+ strain). Don't shake; the DES will
go to the bottom of the tube. After the time requiered, take 0.1 ml from
the top, wash, and grow overnight in 2-5 ml of medium. After that, plate
in selective/screening plates.
To inactivate DES, pour it in hot water and let it cool down.
> Any suggestions? or references?
This protocol came directly from:
S.R. Maloy. (1990) Experimental techniques in bacterial genetics. Jones
and Barlett Pub. Boston, Mass.
There is no DES protocols, but detailed information in other methods, in:
JH Miller. (1992) A short course in bacterial genetics. CSHL press, Cold
Spring Harbor, NY
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