Help! - EMS mutagenesis

["Marianne Leverone ", BIO]

Tim:  I have used 0.6% EMS (vol/vol) in the past on Pseudomonas cultures 
and have gotten good results after an enrichment procedure.  Marianne

On Sun, 9 Jul 1995, Tim Barnett wrote:

> 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
> 
>