UW-Stout/Caffeine FA22

Yeast Caffeine Torture

Caffeine Solubility: 2g/ 100 mL

Pilot Experiment Protocol

Materials:

• Sterile Water
• Caffeine
• Wild Yeast
• Weigh Boat
• Microcentrifuge tubes
• Ethanol spray to sterilize equipment

Stock Solutions

Underneath the hood

1. Make a stock solution at a concentration of 0.1 M

• • Add 0.19419 g of caffeine to 10 mL water
  • Vortex briefly

2. Make a stock solution at a concentration of 0.05 M

• • Take 250 ul of 0.1 M and add 250 ul water
  • Vortex briefly

3. Make a stock solution at a concentration of 0.025 M

• • Take 250 ul of 0.05 M and add 250ul of water
  • Vortex briefly

4. Make a stock solution at a concentration of 0.0125 M

• • Take 250 ul of 0.025 M and add 250 ul water
  • Vortex briefly

5. Make a stock solution at a concentration of 0.00625 M

• • Take 250 ul of 0.0125 and add 250 ul of water
  • Vortex briefly

6. Make a stock solution at a concentration of 0.003125 M

• • Take 250 ul of 0.00625 M and add 250 ul of water
  • Vortex briefly

7. Make a stock solution at a concentration of 0.0015625 M

• • Take 250 ul of 0.003125 M and add 250 ul of water
  • Vortex briefly

8. Make a stock solution at a concentration of 7.8125 x 10^-4 M

• • Take 250 ul of 0.0015625 M and add 250 ul of water
  • Vortex briefly

9. Make a stock solution at a concentration of 3.90625 x 10^-4 M

• • Take 250 ul of 7.8125 x 10^-4 M and add 250 ul of water

10. Make a stock solution at a concentration of 1.953125 x 10^-4 M

• • Take 250 ul of 3.90625 x 10^-4 ul and add 250 ul of water
  • Vortex briefly

Yeast + Wells

1. Vortex the yeast briefly before use.
2. To the first well, add 50 ul of 0.1 M stock solution and 50 ul of yeast.
3. Repeat the first step in the next well for 0.05 M solution, continuing to add each stock solution to the next wells.

Inducing Stress Protocol

Materials

• 10 mL 0.1 M Stock Solution
• Test tubes
• Yeast sample