[Yeast] Problems with KC8
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.
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