UW-Stout/Caffeine 2 SP21

Pilot Experiment 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.

Materials

• 0.22g Caffeine
• Sterile H2O
• Syringes and purifier
• 96 Well assay plate
• Wild type yeast cells

Pilot Doubling time : Td = (146-66) x ln(2)/ln(.6/.2) = 88 minutes

Concentration of solution added to Yeast (uM) { | class=”wikitable” | - ! Concentration of solution added to Yeast (uM) ! Volume of solution added to yeast (ul) ! Water added to the solution (ul) ! Volume of the Stock solution of 100 mM (ul) ! Volume of the Stock solution of 10 mM (ul) | - | 0 | 50 | 50 | - | 0 | - | 0.1 | 50 | 49 | X | 1 | - | 0.25 | 50 | 47.5 | X | 2.5 | - | 0.5 | 50 | 45 | X | 5 | - | 1 | 50 | 40 | X | 10 | - | 2.5 | 50 | 47.5 | 2.5 | X | - | 20 | 50 | 30 | 20 | X | - | 50 | 50 | 0 | 50 | X | }

Caffeine concentration.

1. 0.25
2. 20
3. 0.1
4. 2.5
5. 0.50
6. 50
7. 1
8. 0.0

We chose series 1, the 0.25 concentration of caffeine in yeast, because it has a consistent linear growth curve that has few to no discrepancies. We first chose series 7 because it is in the middle for all the growth curves, but makes the yeast grow to fast resulting in the cells dying. Series 7 was too high of a concentration for the sensitive yeast cells.

Protocol : Stressing the Knockout Strands with Caffeine

Materials

• Sterile H2O
• Yeast Strands
• 96 Well Plate
• Caffeine Stock Solution

Protocol

1. Create enough 10 uM stock solution, mix 47.5 ul water and 2.5 ul stock solution of 10 mM to replicate series 10 (0.25 concentration) to replicate series 4 in the Pilot experiment
2. Add 47.5 ul sterile H2O to each of the wells
3. Add 2.5 ul of diluted caffeine solution to each well
4. Add 50ul of each strand of yeast being tested
5. After solution in the wells is completed put 96 well plate into the SpectraMax Plus 384 Microplate Reader where the data will be collected every 5 minutes for the next 24 hours

Doubling Time

Pilot Doubling time : Td = (146-66) x ln(2)/ln(.6/.2) = 88 minutes Final Experiment Doubling Time : Series 1 and 3 ; Td = (199-1) x ln(2)/ln(.25/.1) = 150 minutes Series 4 and 6 ; Td= (217-1) x ln(2)/ln(.2/.1) Series 2 ; Td = ( 217-103) x ln (2)/ln(.12/.1) Series 5 ; Td= (217-1) x ln(2)/ ln(.1/.09)

1. YOR387C = 150 minutes
2. YKL121W= 433 minutes
3. YBR225W = 150 minutes
4. YNL018C = 216 minutes
5. YLR426W= 1421 minutes
6. YGL138C =216 minutes

We determined that the yeast cells were extremely sensitive to the higher concentrations of the caffeine stock solution. We chose the concentration 0.25 because the cells were less sensitive and did not grow too fast. We were able to slow the growth rate more than half with the 0.25 concentration.