Kill all but spores

Crosses between cells result in asci, but many of the haploid cells will be maintained and also diploids that did not yet sporulate might be hanging around. To assure only spores are maintained, a double treatment can be performed.

Glusulase

Glusulase (NEE154001EA PerkinElmer) is an enzyme mix that digests cell walls of fungi.

1. Scrape cells of a mating plate (EMM-N) and suspend these in 0.5 ml sterile water
2. add 8 ul Glusulase (use the aliquot in the 15ml tube in the door of fridge 1)
3. incubate overnight at 32°C
4. check if the cells are dead
5. spin down the cells and resuspend in water

30% ethanol treatment

In stead of using Glusulase an ethanol treatment can be performed. This is fast and cheap, but will not break the wall of the asci. This is for the lab strain not required, as the spores will be released from the ascus naturally. For other strains, this has not been studied, so be aware of that single spore colonies might not be so single spore.

1. Scrape cells from a mating plate and resuspend in 30% ethanol.
2. incubate at room temperature for 30 minutes
3. spin down the cells at ~1800xg
4. remove supernatant and resuspend cells in 1 ml sterile water

or do both!

Even thought Glusulase does a good job, if only sexual offspring is required, on top of the enzyme treatment the ethanol should be performed.

1. after the ON incubation in glusulase, spin down cells
   1. If you do this, you don't need to spin down cells. You can calculate the amount of 96% ethanol needed to reach a concentration of 30% in the end.
2. resuspend in 30% ethanol
3. continue at step 2 of the ethanol treatment