32P labeling of S. Cerevisiae

Michael J Conboy conboymj at Stanford.EDU
Wed Sep 13 14:09:30 EST 2000


Yeast can have substantial reserves of phosphate (polyphosphate), in the
vacuole, and will not label efficiently until the reserves are used up.  
How long are you growing them in -P media, and it it P-limited or truly
-P?  Before you break out the P-32 again, you might want to passage a
culture for some time in -P medium, until it slows in growth, just to see
how long it takes to become phosphate depleted.  Or, if you have a pilot
culture growing a number of days ahead of your experimental culture, both
in -P medium, then you'll know ahead of time when to switch to P-limited
medium.

Hope this helps,
Mike


In article <8pn31g$dg0$1 at mercury.hgmp.mrc.ac.uk>,
Brian Baker  <brb at chem.ucla.edu> wrote:
>I'm having a hard time trying to label my yeast cells with 32P. the
>label does not seem to be incorporated. I'm using 32P in the form of
>H3PO4, I'm growing my cells in -phosphate media, and I label them for
>~45 min at 30 C. Does anyone know what's I can do? or have a better
>protocol?
>




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