Colony PCR of my propidium iodide stained ADE2 yeast cells ;-)
lichten%bchem.dnet at DXI.NIH.GOV
Fri Sep 30 08:34:51 EST 1994
Re: Various methodological questions
1) FACS prep of yeast. We use a method similar to Fatima's which was
given to us by Andy Hoyt. The only real differences are those of timing.
* Fix in 70% EtOH for 1h @ room temp. Less clumpiness if you
resuspend in water, then add the EtOH.
* Spin, resusp. in 50mM TRIS 7.5, sonicate, add 1mg/ml boiled RNase
and incubate 1hr @ 37 C, overnight in the fridge.
* Spin, resusp. in 5 mg/ml pepsin in 0.05 N HCl (made FRESH).
Incubate 5 min at room temp.
* Spin, resup. in PI staining solution (similar to Fatima's, 180 mM
TRIS, 180 mM NaCl, 70 mM MgCl2, pH 7.5, .05 mg/ml propidium iodide (you can
make a 100x PI stock). Incubate 1hr @ room temp, then in the fridge
I want to thank Andy Hoyt for giving us this procedure.
ALWAYS check your staining using a fluoresence microscope--if you didn't
RNase enough you'll get lots of cytoplasmic staining and yr FACS scans
won't be interpretable. Also, with many machines you will need to use a
diploid strain, although our machine (a BD FacScan) was able to see
haploids. Remember that most FACS techs are used to dealing with mammalian
cells. Remind them of what the genome content of yeast is and have them
adjust their sensitivity accordingly.
2) Bert asked about ADE2 and the ARS. See Storz and Linder, Gene 95:91-98
for a minimal ADE2 construct.
3) Charles asked about PCR with yeast colonies. This was a topic of
considerable discussion in the yeast news group some time back. We have
come up with the following complex procedure (as have many others).
* Grow yeast colonies. Make sure that they are fresh--two days,
not those grungy old plates from the back of your bench.
* Pick up about 1/4 - 1/3 of a yeast colony. Put it in a standard
PCR reaction. Mix it up.
* Run the PCR reaction.
lichten at helix.nih.gov
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