Transformation after EMS

S L Forsburg notmyaddress at hotmail.com
Mon Feb 7 16:16:35 EST 2000


> 
> I would like to mutagenize yeast and screen the mutants that are
> resistant to ribosome toxin expressed by a plasmid. I prefer not to put
> the plasmid into yeast before treatment of EMS since I will potentially
> get a lot plasmid bourne mutants. But I am not sure if yeast after
> treatment of EMS could take DNA by LiOAC transformation. Appreciate any
> suggestion.

I would not do this.  

First of all, only a fraction of cells in any culture are actually
transformed .  That means that you will lose a lot of your mutant pool
even before screening, because they won't take up the plasmid.  This
defeats the purpose of the screen, which is to screen as many mutants as possible.

Second, the yeast probably contains multiple copies of the plasmid (if
2u),  which offers a much smaller target than the genome for mutation. 

It's much easier to get potential positives first and then do a
secondary screen, perhaps by plasmid shuffle, to make sure the plasmid
is intact and the phenotype is true.  That way you can be confident you
are screening a respresentative selection of your collection of mutants.

Just a thought from an old yeaster.


-- 
-susan
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S L Forsburg, PhD  forsburg at salk.edu
Molecular Biology and Virology Lab          
The Salk Institute, La Jolla CA 
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