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Yeast Sporulation (liquid)

Updated 11/24/2022 08:11 AM
Step-by-step
by AcubeSAT (created by Asterios Arampatzis)

Introduction

Making Schwarzenegger spores

Materials

  • Presporulation medium
    • 10g/L Yeast Extract
    • 20g/L Peptone
    • 10g/L Potassium Acetate
  • Sporulation medium
    • 10g/L Potassium Acetate
  • Falcon Tubes (Or Double-Lock Tubes)

    Procedure

    1. Pick a colony or inoculate 100µL of liquid culture diploid cells in 5mL of YPD (SD-His-Met ?) medium in a 15mL falcon tube
    1. Incubate overnight at 30oC in a shaking incubator at 140 rpm for 18-20 hrs
    1. Pick 200µL of the YPD (SD-His-Met ?) diploid cultures and inoculate at 5mL of fresh YPD (SD-His-Met ?)
    1. Incubate at 30 oC in shacking incubator until the cultures reach the concentration of 1–2 × 107 cells/ml or OD=1 (it takes around 5-6 hours)
    1. Centrifuge at 3000 rpm for 5 min
    1. Discard supernatant
    1. Resuspend cells in 5mL of pre-sporulation medium and allow to grow for 18-24 hours at 30oC in shacking incubator
    1. Centrifuge at 3000 rpm for 5 min
    1. Discard supernatant
    1. Resuspend in 5mL of sporulation medium and allow to sporulate for 72 hours at 22oC in shacking incubator

    Notes

    - During the pre-sporulation and sporulation steps it is important for the cultures to grow under adequate ventilation

    - If the case is using flasks instead of falcons, it is recommended to work under sterile conditions using the burner

    - If you want to accelerate the sporulation process you can move the sporulation cultures at 30 oC after one day of incubation at RT

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