Plasmid prep for yeast two hybrid?

neale at mbcf.stjude.org neale at mbcf.stjude.org
Fri Feb 24 13:00:44 EST 1995


I apologize for this somewhat trivial question, but............


Someone posted about a month ago (and I can't find it in the archives) the
minimum volumes of Solutions I, II, III for alkaline lysis of bacterial cells
for large scale plasmid preps. I think the volume mentioned was 5 ml per 100 ml
of culture volume.

I don't know if others using the yeast two hybrid system encounter this problem
also, but we find that when you plate out your bacteria to prepare the plasmid
library for transformation in yeast you get very inefficient recovery of
plasmid, and poor lysis of bacterial cells using the "recommended" protocols.
You end up with about 4 liters of stationery cells in Terrific Broth, which is
not all that easy to manipulate in the normal lab setting.

So if anyone has had better succes in purifying the plasmid library, please let
me know.

Thanks, 

Geoff Neale



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