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 M 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.

Stock Solutions

Based off of this data, we decided to use the C4 stock solution moving forward since this concentration had a noticeable effect on the cells, but was not too concentrated that it killed them.

Inducing Stress Protocol

Materials

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

Stock Solution

1. Make 1 ml of stock solution at a concentration of 0.0125 M

• Add 125ul of the 10 ml 0.1M stock solution to 875ul of sterile water
• Vortex Briefly

Yeast + Wells

1. Vortex the yeast briefly before use.

2. Add 50ul of the 0.0125 M stock solution and 50ul of wild type yeast to the first well.

3. Repeat for each knockout gene in the subsequent wells.

Trial 1

Trial 2