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help! - EMS mutagenesis

Tim Barnett T.Barnett at path.utas.edu.au
Sat Jul 8 21:38:10 EST 1995

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

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