McClean: Sporulation

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 day (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

1. 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).

1. Pellet cells, wash in 1 mL of 1% potassium acetate, and resuspend in 3-4 mL of 1%

potassium acetate.

1. 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).
2. Check culture for spore formation on day 2 and continue checking until you see tetrads.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. *Megan N McClean 13:25, 30 September 2011 (EDT): According to the Botstein lab, 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). You may need to play with the concentration of nutrient that you add. So far, the McClean lab has only sporulated prototrophic strains using this protocol, so the first person to try it/get it to work with an auxotrophic strain, please leave a note here!
2. *Megan N McClean 13:25, 30 September 2011 (EDT): To sporulate yeast cells need to be starved for nitrogen in the presence of a nonfermentable carbon source.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Adapted from the Botstein lab protocol available at: http://www.princeton.edu/genomics/botstein/protocols/Sporulation_and_Tetrad_Dissection.pdf

Contact

*Megan N McClean 13:29, 30 September 2011 (EDT) or instead, discuss this protocol.