Two Hybrid Sceening Problem

Shin Enomoto shin at
Sun Apr 9 09:14:55 EST 1995

: Hello Wim,

: what about you screen them after your transformation on SD -W -L -H +3AT
: and then grid the upcoming colonies onto SD -W -L to get colonies big enough
: for the filter assay?

What about replica plating or picking the intial positives from SDC -W -L 
-H +AT onto SDC -H or SDC -H+3AT.  The first plating should have 
elminated simple HIS+ guys and thus subsequent expression of the HIS+ 
should all require the presence of the 2 plasmids so a selection for HIS 
protorophy should be sufficient.  
The other thing is to make sure plates are not drying out.  Five days is 
a long time and plates yeast simply quits growing when plates get too dry.

Hope this helps

: >In using CG-1945 for a two hybrid screening I'm having trouble in obtaining
: >colonies big enough to lift (for the lysis in liquid nitrogen). The colonies 
: >start growing after 2-3 days but stop after 5 days. How can I overcome this ? 
: >e.g. changing X-gal assay, or using different plating techniques (I'm using SD 
: >plates lacking LEU,TRP & HIS, 5 mM 3-TA). All suggestions are welcome.
: >

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