[Yeast] Problems with KC8

RAUL GONZALEZ raulgg32 at hotmail.com
Thu Dec 1 06:05:31 EST 2005


I have retrieved positive Leu+LacZ+ clones from a library screening and now 
I'm trying to isolate the library plasmids using KC8 electrocompetent cells 
to rescue it.  I'm using the standard Yeast plasmid purification procedure.  
I'm plating the transformed colonies in M9 media plus the -Trp drop out(as 
well as Amp-100ìg/ml & Kan-30ìg/ml) media. Colonies appear after an 
overnight incubation at 37°C(Clontech states it should take from 36-48h) . 
Then I miniprep using LB amp/kan for the overnight liquid culture (they do 
not grow in liquid M9 media).   Unfortunately, I'm obtaining  (100-300 
colonies) just the bait or the LacZ reporter plasmids during the isolation 
process and only on one occassion I obtained the library plasmid. Could 
anyone tell me why I have such a high background from the other plasmids.

Thanks

Raul Gonzalez




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