Im currently using the Clontech 2-hybrid system Mk II. I have my bait
cloned into pAS2-1 and have tested this construct in strain Y190 for
autonomous activation of the his+ and B-gal genes which fortunately
doesnt happen so I am ready to try the screen for real.
What I would like to do is the Sequential transformation,
ie. grow up Y190+bait and then transform with the AD library, this
should increase the efficiency over the simultaneous transformation and
therefore I should have to plate out less transformants. However, this
requires that I grow up my Y190+bait in SD/-trp to produce a stationary
phase culture and then add to YPD for 3hrs and then use this culture to
create the competent cells for the transformation.
Clontech say that this is fine providing the bait is not toxic to
the yeast, thereby killing them or inhibiting their growth. How does one
tell if this is the case? If the cells die, I can spot that (!) but
how does one gauge if they are growing too slowly?
My transformed Y190's seem to grow fine on SD/-trp plates though
nowhere near as fast as wtY190 grows on YPD but I believe this is to be
expected. I tried to grow up 50ml of the transformed yeast in liquid
SD/-trp but even after 2days at 30C the culture was nowhere near
Is this to be expected - are they growing slowly simply because they are
in SD media, or is it because my bait is somehow inhibiting the growth?
Should I just wait longer for the culture to reach stationary phase,
or go with the simultaneous transformation?
I would be interested to hear which approach other people have taken to this
problem (simutaneous vs sequential transformation) and how long strains
usually take to grow in SD media.
Thanks in advance for any help you can give!
Dr. Simon Twigger,
Medical College of Wisconsin, Milkwaukee. WI