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
- Make a 100μM stock solution of caffeine and H2O.
- Take 0.1942 grams of caffeine to 10ml of H2O to form a stock solution of 100C.
- Make another stock solution of 10μM of caffeine and H2O.
- Dilute the 100μM stock solution of 10μM stock solution by adding 4μl of 100μM of the stock solution and 36μl sterile H20.
- Filter the caffeine solution using the syringes and purifier then put in microfuge tubes, freeze at -20°C.
- Dilute the 100μM stock solution into the 12 concentrations using the table below.
- Fill the wells with different concentrations of caffeine solution.
- 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.
- Once results are computed the data curve should be put on the growth data curve.
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 | - |
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 |