Maheshri:Mating

From OpenWetWare

Revision as of 20:22, 7 September 2009 by T.l. To (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Image:Banner01.gif

Home        Research        Lab Members        Publications        Software        Internal       


Non-selective Mating of Yeast

Leah Octavio

6/26/08

Day 1

  1. Grow overnight 2ml cultures of a and alpha haploid yeast (in YPD or selective media).

Day 2

  1. In fresh YPD (or selective media, if strains have plasmids), inoculate with roughly 50:50 cell ratio of each haploid from overnight culture. Inoculate to starting OD600 ~0.05 to 0.1.
  2. Grow in 30C roller drum for 8-12 hrs to let the a and alpha cells mate. It’s best to start mating the cells either at the beginning of the day and plate before leaving. Alternatively, if it’s already late in the evening, you can also grow them overnight and plate first thing in the morning.
  3. (Optional step) If the two strains being mated have different colored fluorescent proteins that are expressed in regular media conditions, before plating the mated cells, you may take a quick look at the culture using the microscope to estimate how much of the cell population is diploid by looking at the appropriate fluorescence channels. You may choose to grow the cells longer if only a few cells are diploids. Also, if your cells don’t have any fluorescent reporters, you could check roughly for mating success by looking at the cells’ sizes; diploids on average are about twice as large than haploids. The fraction of diploids you observe in the sample will give you an idea of how many colonies you should pick later when screening for diploids.
  4. Make two 100-fold serial dilutions of the culture, and plate 150-200 ul of each (undiluted culture, 1/100, 1/10000). You may dilute in sterile water or liquid media. If your cells tend to flocculate, sonicate the cells (use either the sonicator in the Prather lab or the Cooney lab) before plating. It is also general good practice to sonicate the cells prior to plating to ensure that colonies arise only from single cells, and to prevent haploid contamination in the diploid colony that you will pick later. Use either YPD or selective plates. Place in 30C incubator 1-2 days (colonies should be visible by the next day, if plating on YPD).

Day 3 or 4 (depending on whether colonies are large enough on the plates)

  1. Choose plate(s) with single colonies visible.
  2. For cells with different colored fluorescence reporters in the haploids: select 10-20 colonies on the plate, and mark each colony on the plate. Then, add 1.8ul water on a slide, and take a toothpick and place about ½ colony of cells onto the drop. Swirl toothpick around to mix the cells with the water before placing on coverslip. View cells in microscope and use fluorescent reporter expression to screen for diploids. Once you find the diploid, streak the rest of remaining colony from the plate to a new, fresh plate and verify that it’s a diploid using check primers for the MAT loci (available from my box or Tek Hyung).
  3. For cells with no fluorescent proteins (or same colored fluorescent reporters) in the haploids: select 10-20 colonies on the plate. Pick off colony using toothpick and patch onto a new fresh plate. Grow overnight 30C. The next day, you may either check PCR all the strains (using MAT loci primers) to look for a diploid. Alternatively, you may take a look at the cells under the microscope first to look for generally large looking cells that could be potential diploids. After picking which cells look large, you may just do a check PCR on these subset of colonies instead of all the colonies you picked.

Final notes:

To save one day on the mating assay, I have also tried “quick-and-dirty” mating where instead of growing overnight cultures of the haploids on the first day, I simply mix a stick full of each haploid from a plate into 2ml liquid culture, and grow for 8 hrs before plating. If you do this in the morning of Day 1, and then at the end of the day, you may plate the cells and then screen for diploids the following day (usually, the colonies are still quite small in the morning but are large enough by the end of the day).

I originally mated yeast at room temperature using the orbital shaker, so that the cells wouldn’t grow too fast. Unfortunately, the shaker sometimes falls off the bench and the culture spills out of the tube. Mating in the 30C incubator roller drum works just as well.

Possible problems with the mating could occur if the cells are plated too early or if they are overgrown. Also check that your strain has no mating defects. If your strain has a particularly low mating efficiency, you may need to use selective mating to obtain a diploid.

Personal tools