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pichia and 96-well plates

Alex Beeser A_Beeser at fccc.edu
Thu Jul 26 09:16:00 EST 2001

  As I have to screen several clones in both mut+/Muts backgrounds in a
variety of growth media
( MM, BMM, BMMY) I was hoping to be able to do the screening for best
expressing clones en masse.  As I'm expressing the proteins off the AOX1
promoter and secreted by the cerevisiae alpha factor secretion signal
this seems problematic.  My biggest concern is aeration followed closely
by temperature control.  Has anoyone tried to induce multiple clones in
multi well plates ?  If so how did it go ?  The alternative is to use
lots and lots of flasks and space in every available shaker on the floor
for upto a week which won't endear me to my coleagues.  I would probably
induce the entire plate and remove aliquots of the supernatant and do
dot-blot westerns as a function of time.  Positive clones would then be
subjected to sds-page and westerns.

  Also has anyone had dramtic improvement in expression by using
"jackpot" integration clones ?
The selectable marker is zeocin, the manual says that you can go upto 2
mg/ml ( which is 20X) zeocin in order to isolate multiple integrants.
Zeocin however is quite expensive and I can't rationalise why 20X Zeo-R
would be dramatically better than say 5 to 10 X.

Tanks in advance,

Sandy Beeser

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