Sporic meiosis

Sporulation is how you generate haploid yeast strains from diploid yeast strains. An example of this would be to generate haploid deletion strains from a diploid strain from the het dip (heterozygous diploid) deletion collection.

There are several different protocols for sporulation, both outside our lab and within it. This is the one I learned from Brett and Kim, who learned it from Kristan. It works great for me so far but YMMV. If this isn't doing it for you, try GNA Sporulation which takes longer but is reported to be more reliable.

You'll need: Culture Tubes, YEP, 50% Glucose Stock, Toothpicks, Sporulation Media, 1000ul tips, your diploid strain(s), and 6-8 days' use of the 30C Shaker.

Note: Steps 1, 2 & 3 may already be done; if so, proceed to step 4 & 5

1. You'll need YEP + 2% glucose, which can be made from lab stock YEP and 50% Glucose Stock solutions if you're out. This stuff is very nutritious to wee beasties, so keep it closed up tight if you want it to stay sterile for long. If it's at all cloudy, it's contaminated; make or get more.

2. Add 3ml of YEP + 2% Glucose to a glass culture tube for each strain you will be sporulating, and label the lid and tube with a piece of lab tape or a tough spot.

3. Inoculate each tube using a toothpick from a single colony of your diploid strain in question. Shake at 30C overnight.

4. The next day, add 3ml of SPM (sporulation media) to a new culture tube for each strain (be sure to label your new tubes).

5. Using 1000ul tips and a P1000 pipettor, for each strain, aliquot 300ul of your diploid culture into the new tube containing 3ml of Sporulation Media. Shake at 30C for 5-7 days before going to Tetrad Dissection