limited growth on sd

Carol Berkower carolb at yam-suff.cc.huji.ac.il
Tue Nov 14 14:14:03 EST 1995


In article <47qq90$7d0 at decaxp.harvard.edu>, ddeaguia at oeb.harvard.edu (Dan
Deaguiar) wrote:

> Hi all, I have recently begone wotking with yeast and I'm curious to know
> whether it is normal for them to grow to a limited size on sd media. I
> plate cells on sd media suplemmented with all aa's except the ones of
> interest and upon transformation i do obtain colonies. The problem is
> that after 5-6 days, the colonies reach 1-1.5mm in diameter and stop
> growing. This also occurs when I plate cells on sd supplemented with all
> aa's. Is this normal? Is there a problem with my media? I am making it
> according to Sherman's recipe...Might this be strain dependant (I am
> currently using one strain only)? Any insights would be appreciated.
> 
> Thanx,
> 
> Dan D.

Hi Dan, I've never replied to one of these before, but your question was so
close to my heart I couldn't resist.    First of all, I am assuming that
you are working with S. cerevisiae.  If so, it's unusual for strains to
take 5-6 days to reach a decent colony size on either SC or SD.  Two or
three days is normal, unless you've got a slow-growing mutant.  I would be
suspicious of anything that took 5 days to creep up.  Also, yes, strain
differences are *extremely* important with yeast.  You don't know if the
problem is in your plates or in your cells - you really need to compare
several strains, and isogenic strains with several auxotrophies, to get a
handle on things.    But the real reason I responded is to pass on the
following tale, which may be pertinent.  Sorry it's so long-winded.

Several years ago I TA'd in the yeast genetics course at Cold Spring Harbor
and we ran into a problem where all our Leu- auxotrophs (specifically) grew
poorly on SD dropout mix.  It turned out it was because we had combined two
recipes - the one for SD with all-but-one (or several) amino acids (termed
SC-aa, eg. SC-Ura, for "synthetic complete"), and the one for SD lacking
all amino acids except for the few that were specifically added (SD+aa, for
"synthetic dextrose").

Our problem was that Leu- auxotrophs didn't grow well on SC-URA, SC-TRP,
etc. plates.  What we had done wrong was this: in order to simplify our
production of plate media (of which there were 20+ varieties), we made one
dropout mix to add to all the SD flasks post-autoclaving.  ie., rather than
separate bottles of 10X dropout mix for -Ura, -Trp, -Leu, etc. plates, we
made one 10X -Ura-Leu-Met-Trp etc. mix and added that to all our SD plate
media.  Then we would add back the other amino acids from individual
stocks.  The problem was that we determined concentrations as follows: we
used an SC- recipe for the 10X mix and an SD+ recipe for the add-backs. 
But it turns out that when Leucine is combined with many other amino acids
in the (SC-) medium, it is not as effective as when it is alone or one of
only a few additions, ie., in the SD+ medium.  In other words, if you look
at the recipes for SC and SD mix in the CSH Yeast Methods Book, you'll
notice a 3x higher concentration of Leucine required in the SC mix than in
the SD mix.  So your problem could be, perhaps, insufficient Leucine.

OK sorry that was such a long explanation, but I've seen the problem creep
up elsewhere.  Obviously this is not your problem if your strains are not
Leu auxotrophs - you could check by comparing Leu+ and Leu- strains on the
same plates.  If that doesn't work, I would try spreading extra Trp on one
plate, since this can go bad, and comparing growth on plates with and
without extra Trp (if you've got a Trp auxotroph).  But really you should
try spreading a few well-characterized, isogenic strains with different
auxotrophies on your plates and see what does or does not pop up.  But
aren't there plenty of people at Harvard who can help you work this out?

Good luck - Carol



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