Two Hybrid Sceening Problem
shin at cis.umn.edu
Sun Apr 9 09:14:55 EST 1995
DSCHLIEP at GENETIK.UNI-KOELN.DE wrote:
: 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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