McClean: Yeast Mating Halo Assay
|Line 29:||Line 29:|
mating type, the cells arrest.
mating type, the cells arrest.
Revision as of 14:42, 15 March 2013
This is a protocol for checking the mating type of a strain of interest using tester strains that are super-sensitive to mating pheromones. Your strain of interest is patched (or stamped or replica plated) onto a sensitive tester lawn and the formation of halos on the lawn (due to growth inhibition of the sensitive strain) indicates that your strain is the opposite mating type of the tester.
- 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
- YPD + 0.05% Triton X-100 (see Notes below)
- 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.
Elia, L. and Marsh, L. (1996) Role of the ABC transporter Ste6 in cell fusion during yeast conjugation J Cell Biol 135 741-751
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.
- Megan N McClean 14:37, 23 September 2012 (EDT) The yMM422 and 421 strains both showed good halos for me this time. Here is what I did (granted, this was very sloppy because I was in a rush, so I am surprised it worked as well as it did): I took several colonies of yMM422 and yMM421 off of a very old YPD plate they had been struck onto and put them into about 1ml of liquid YPD. I picked the lightest red colonies. I'm guessing just from the shade of the YPD after I had mixed the colonies into it that the OD600 was around 0.8-1. I immediately took 200μL of this and spread it using glass beads onto a YPD plate. This I allowed to dry for 30 minutes at 30°C. Once it was dry I frogged the tetrads onto it, and left it for ~24 hours growing at 30°C. One of the yMM422 plates worked well (the drier one) and the other one that didn't quite dry before I frogged onto it only shows a few clear halos...perhaps because there are some places where the yMM422 lawn is too thick?
- Megan N McClean 15:40, 15 March 2013 (EDT) Jue Wang (Springer Lab, Harvard Medical School) has had good success getting clear halos using YPD plates with 0.05% Triton X-100 and our yMM422 tester strain. He found that there is almost no halo when the test is done on regular YPD, however on YPD + 0.05% Triton X-100 he reproducibly gets good halos (the Triton X-100 is an important component as pointed out in Elia and Marsh, 1996). He has been able to get the protocol to work without being too careful about the yMM422 growth conditions. Apparently all of the following have worked: 300μL of saturated yMM422 (in YPD) sitting at RT for 4 days plated with beads on a square plate (see images), testers grown for 1 overnight and plated with beads, and scraping testers off of a plate and diluting them in water at some unknown concentration and making a lawn with glass beads.
- Megan N McClean 14:01, 03 November 11(EDT)
or instead, discuss this protocol.