Crossing two strains

Crossing two heterothallic strains from plate

If both strains are not switching and of opposite mating type and grown on plates, crossing two strains is fairly straight forward.

• Make a mating plate with the correct supplements,
• Add a dollop of cells from strain 1 with a toothpick onto the plate,
• Add on top of those cells a dollop of cells from strain 2 and,
• Mix the two types of cells by carefully stirring (don't break the agar).
• On one plate a large number (up to 64) of crosses can be made if needed.
• Incubate the plate upside down at 25°C for 4 days (use parafilm or a bag to avoid drying out)
• To test if the mating was successful, test for asci under the microscope and continue with glusulase and/or ethanol treatment.

Crossing two heterothallic strains in liquid

If both strains are not switching and of opposite mating type and grown in liquid, crossing two strains is almost identical to above.

• Make a mating plate with the correct supplements,
• Place a small droplet (~2-5 μl) of strain 1 on the plate,
• Add on top of that droplet an equal volume of cells from strain 2,
• Wait for the liquid to be absorbed into the plate/evaporate,
• On one plate a large number (up to 64) of crosses can be made if needed.
• Incubate the plate upside down at 25°C for 4 days (use parafilm or a bag to avoid drying out).
• To test if the mating was successful, test for asci under the microscope and continue with glusulase and/or ethanol treatment.

Forcing outcrossing of one or two homothallic strains

When one or both of the strains is homothallic there are two options, forcing outcrossing based on 1) auxotrophic/resistance markers or, 2) when selective markers make this possible the outcrossed offspring can be selected in a Random spore analysis.
For forcing outcrossing what is required is that the two strains should have markers that can complement each other in the diploid zygote in an environment where neither of the individual strains can grow. Most common ade6-M210 with ade6-M216.
Also see: Creating diploids
Common used combinations are for example:

• his- leu+ with his+ leu- grown on minimal medium without supplements
• ade6-M210 with ade6-M216 grown on minimal medium without supplements
• natMX with kanMX grown on YES with Nourseothricin AND G418

Procedure

• Setup a cross with two strains from liquid (use high density) or plate as described above
• Incubate this plate for 16 to 24 hours
• With a toothpick take cells from the cross, and streak these out on the double selection plate
• Incubate at 32°C for 2-3 days
• Pick colonies from the double selection plate and streak out on YES + Phloxin B

During the incubation, the haploid strains will not be able to grow by themselves, and only the diploids will be able to survive. Generally, diploids are very short lived, as these immediately go into meiosis and sporulation, which stops their growth. Rich medium induces diploid cells to continue growth and the increased temperature suppresses meiosis, which should result in a limited number of diploid colonies.
Phloxin B and flowcytometry can be used to test for ploidy. The diploid colony should contain a copy of each of the haploid parents.

• From the YES+Phloxin B plates, pick the dark pink colonies (diploid colonies are darker because dead cells accumulate the pink color).
• Transfer the diploid cells to a fresh mating plate and incubate at 25°C to induce meiosis.
• The asci (called 'azygotic' as these are formed not directly from the zygote, but from diploid cells) that are now formed contain spores that should be outcrossed.