I am trying to mutate an endogenous alkaline phosphatase gene from
Aeromonas sobria and intend to perform subsequent TnphoA mutagenesis on
this strain. I have screened over 5000 survivors following growth for 30
min. in EMS at concentrations varying from 0.8% to 6.4% v/v. Is the right
range or am I too high/low?
My protocol involves resuspending a log phase culture in saline, adding
EMS and incobating for 30 min. at 37C. Following this incubation I
resuspend in fresh saline and plate onto LB plates. Alkaline phosphatase
mutants are identified by growth as white colonies on LB/BCIP plates.
Thanks in advance,
Tim Barnett
