classical mutagenesis? the best way to do it

[Rafael Maldonado]

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
> results.

Christian,

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


Rafa

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