Kill all but spores

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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 or MMX

Glusulase (NEE154001EA PerkinElmer) is an enzyme mix that digests cell walls of fungi. It works really well, but is also rather expensive (~30 EUR/ml). As an alternative you can use Lallzyme MMX, which is a commercially available enzyme mixture used in wine making that has the same features, works really well and costs about 60EUR/liter. If possible, use MMX.

  1. Scrape cells of a mating plate (EMM-lowN) and suspend these in:
    1. 150µl water with 100 mg/ml MMX
    2. 150 μl sterile water with 8 μl Glusulase (use the aliquot in the 15ml tube in the door of fridge 1)
  2. incubate for at least 2h or preferably overnight at 32°C
  3. check if the cells are dead
  4. spin down the cells and re-suspend in water
  5. Plate out in appropriate dilution on YES to ~100-5000 spores/plate

30% ethanol treatment

Instead of using Glusulase or MMX 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 re-suspend cells in 1 ml sterile water
  5. Plate out in appropriate dilution on YES to ~100-5000 spores/plate

or do both!

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

  1. First do the MMX or the Glusulase treatment as described above.
  2. Incubate in 30% ethanol for 30 min doing:
    1. Add 96% ethanol; you can calculate the amount of 96% ethanol needed to reach a concentration of 30% in the end (~70μl 96% when using 150μl water or 230μl for 0.5ml water)
    2. OR after the incubation in Glusulase, spin down cells, remove supernatant and re-suspend in 30% ethanol
  3. continue at step 3 of the ethanol treatment