UW-Stout/Caffeine FA19

Caffeine Dilution and Concentration of Caffeine Protocol

Materials

• 0.22g Caffeine
• Sterile H2O
• Syringes and purifier
• 96 Well assay plate

Procedure

1. Make a 100μM stock solution of caffeine and H2O.
2. Take 0.1942 grams of caffeine to 10ml of H2O to form a stock solution of 100C.
3. Make another stock solution of 10μM of caffeine and H2O.
4. Dilute the 100μM stock solution of 10μM stock solution by adding 4μl of 100μM of the stock solution and 36μl sterile H20.
5. Filter the caffeine solution using the syringes and purifier then put in microfuge tubes, freeze at -20°C.
6. Dilute the 100μM stock solution into the 12 concentrations using the table below.
7. Fill the wells with different concentrations of caffeine solution.
8. After the wells are completed, place the 96 Well assay plate into the SpectraMax Plus 384 Microplate Reader. Data will be collected every 5 minutes for the next 24 hours.
9. Once results are computed the data curve should be put on the growth data curve.

Concentrations of Caffeine to Yeast Cells

Concentration of solution added to yeast (μM)	Volume of solution added to yeast (μl)	Water added to the solution (μl)	Volume of the Stock solution of 100mM (μl)	Volume of the Stock solution of 10mM (μl)
0	50	50	-	0
0.10	50	49	-	0
0.25	50	47.5	-	2.5
0.50	50	45	-	5
1.0	50	40	-	10
2.5	50	47.5	2.5	-
5.0	50	45	5	-
8.0	50	42	8	-
10	50	40	10	-
15	50	35	15	-
20	50	30	20	-
50	50	0	50	-

Caffeine Concentration in Graph:

Series	Concentration in μM
1	50
2	20
3	15
4	10
5	8.0
6	5.0
7	2.5
8	1.0
9	0.50
10	0.25
11	0.10
12	0.0