UW-Stout/Caffeine SP22

Materials: • Caffeine • Sterile H2O • Wild-Type Yeast Cells • Modified Yeast Cells

Equipment: • Large Test Tube • 1.7 ml Microcentrifuge Tube • 200 ul Pipette • Micro Pipette Tips • Well Plates

Calibration Protocol:

1. Create a 32 mM Solution of Caffeine in Sterile H2O.
2. Set Well Plate with 50 ul of Wild-Type Yeast Cells and the following concentrations of Caffeine.

	Well 1	Well 2	Well 3	Well 4	Well 5	Well 6	Well 7	Well 8
Caffeine Concentration	0 mM	4 mM	8 mM	12 mM	16 mM	20 mM	28 mM	32 mM

As can be seen in the graph above, Well 1 (our control group) shows the expected growth curve. Every group with a concentration of at least 8mM showed little growth and can be assumed dead. Well 2, at 4mM, shows a reduction in growth but not a visible result for future tests.

Knockout Protocol:

1. Create a 4 mM Solution in Sterile H2O.
2. Set Well Plates with 50 uL of each Yeast group of the following:
   1. BY4735
   2. YDL109C
   3. YGL140C
   4. YOR111W
   5. YHL029C
   6. YDR307W
   7. YNL058C
   8. YCL049C
   9. YGR079W
   10. YBL113C
3. Add 50 uL of 4 mM Caffeine Solution to each Well.
4. Incubate