Difference between revisions of "McClean:Phluorin Calibration"
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Miesenbock, G; De Angelis, DA; and JE Rothman (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins ''Nature''''' 394''' 192-195
Orij, R; Postmus, J; Beek, A; Brul, S; and G. Smits (2009) ''In vivo'' measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in ''Saccaromyces cerevisiae'' reveals a relation between intracellular pH and growth ''Microbiology'' '''155''' 268-278
Revision as of 07:18, 5 June 2012
This protocol is for calibrating the ratio of
- Strains of interest to check for mating type
- yMM421 (aka DBY7730, RC634a) MATa ade2 his6 met1 ura1 can1 cyh1 rme sst1-3
- yMM422 (aka DBY7442, XT1-20A) MATalpha leu- ura- ade- sst2
- YPD plates
- Let streaks grow 2 days 30°C.
- Inoculate overnight YPD cultures of the mating type testers.
- Dilute the overnights 1:10 with sterile media or water.
- Spread ~200 μl lawn on YPD plates, one for each mating type.
- Incubate 30°C for 30 minutes.
- Pin your strains of interest to the lawns, or use a toothpick to make small, well-separated patches on the lawn. Make sure to flame the frogger between each plate, or use a fresh toothpick for each plate. Do NOT pin onto tester plates thate are still wet. This will just cause your patches to run all over the plate.
- Incubate 30°C overnight.
- Score whether or not the patch has a halo of space around it. If it does, that means that the lawn strain responded to the pheromone emitted by the patch, and thus that they are of opposite mating type. So, if a halo formed around the patched strain on the a tester plate, the strain itself is alpha. The nice thing about having both the a and the alpha tester plates is that you can double-check your scoring by making sure there is a halo on only one tester plate.
The strains were made in the Thorner lab. See Julius et al (1983) Cell, 32, 839-52 for documentation of DBY7730. DBY7442's exact genotype is unknown. The sst mutations make them super-sensitive to pheromone. When exposed to pheromone of the opposite mating type, the cells arrest.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
- Megan N McClean 11:02, 3 November 2011 (EDT) We have been having some trouble lately with the yMM422 strain. Strains of the opposite mating type have not been forming scorable halos. We may want to try fresh stock from the Botstein lab's -80°C stock. Alternatively, it seems to work 'sometimes' and since none of us are that careful about how many cells we seed or what growth phase they are in when we do so, perhaps we should play around with that and then be consistent in the future.
- Miesenbock, G; De Angelis, DA; and JE Rothman (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins Nature 394 192-195
- Orij, R; Postmus, J; Beek, A; Brul, S; and G. Smits (2009) In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccaromyces cerevisiae reveals a relation between intracellular pH and growth Microbiology 155 268-278
- Megan N McClean 14:01, 03 November 11(EDT)
or instead, discuss this protocol.