Overview
To sporulate yeast cells need to be starved for nitrogen in the presence of a nonfermentable carbon source. This protocol uses acetate as the carbon source.
Materials
- 1% Potassium Acetate (autoclaved)
SPORULATION Mark Hickman uses this protocol and regularly has sporulation efficiencies of >50%. 1. Grow a 5 mL overnight culture; dilute 1:50 in the morning and grow for 4 hours at 30°C (to log phase). 2. Pellet cells, wash in 1 mL of 1% potassium acetate, and resuspend in 3-4 mL of 1% potassium acetate. 3. Incubate at room temperature on a roller wheel for 3 days (can incubate longer, if desired). NOTE: When sporulating, if diploid is homozygous for an auxotrophic mutation, add that nutrient to the potassium acetate (about ¼ the amount listed for addition to SD media… any more and it could be used as a nitrogen source).
Stock Solutions
1% Potassium Acetate
Protocol
- Grow a 5 mL overnight culture; dilute 1:50 in 5 mL in the morning and grow for 4 hours at 30°C (to log
phase).
- Pellet cells, wash in 1 mL of 1% potassium acetate, and resuspend in 3-4 mL of 1%
potassium acetate.
- Incubate at room temperature on a roller wheel for 1 day and then transfer to 30°C for 2 more days (can incubate longer, if desired).
- Check culture for spore formation on day 2 and continue checking until you see tetrads.
References
- Adapted from the Botstein lab protocol available at: http://www.princeton.edu/genomics/botstein/protocols/Sporulation_and_Tetrad_Dissection.pdf
- Elrod S.L., Chen S.M., Schwartz K., Shuster E.O. (2009) Optimizing Sporulation Conditions for Different Saccharomyces cerevisiae Strain Backgrounds. In: Keeney S. (eds) Meiosis. Methods in Molecular Biology (Methods and Protocols), vol 557. Humana Press.