Re concentration of EMS

[pattonaj%phvax.dnet at sb.com]

Rich

in the dim and distant past I used EMS to mutate E.coli and this is the
method that I used. I hope it helps

Harvest cells from 200ml overnight culture
Resuspend in 20ml 100mM Na/K phosphate buffer pH 7.0
Add 200 microlitres EMS (Sigma). Works out to be approx 90mM.
Inc. 37 degrees C for 1 hour with gentle shaking.
Collect cells from 1 ml.
Resuspend in 100mM phosphate buffer and plate out.

I'm not sure if the conc. of EMS was optimal, you may have to titrate it
for your strain of E.coli (I was using a mutant of K12) but it worked.

Best of luck

Amanda