Difference between revisions of "UW-Stout/Caffeine FA22"
(→Inducing Stress Protocol) |
(→Yeast + Wells) |
||
| (20 intermediate revisions by the same user not shown) | |||
| Line 60: | Line 60: | ||
# To the first well, add 50 ul of 0.1 M stock solution and 50 ul of yeast. | # To the first well, add 50 ul of 0.1 M stock solution and 50 ul of yeast. | ||
# Repeat the first step in the next well for 0.05 M solution, continuing to add each stock solution to the next wells. | # Repeat the first step in the next well for 0.05 M solution, continuing to add each stock solution to the next wells. | ||
| + | |||
| + | [[File: OD_600_original_trial_Viv.png|left|thumb|100pxs|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== | ==Inducing Stress Protocol== | ||
| Line 85: | Line 105: | ||
3. Repeat for each knockout gene in the subsequent wells. | 3. Repeat for each knockout gene in the subsequent wells. | ||
| − | [[Image:Knockout_v._caffeine_trial1_Viv.png]] | + | [[Image:Knockout_v._caffeine_trial1_Viv.png|left|thumb|100pxs|Trial 1]] |
| + | |||
| + | [[Image:Knockout_gene_v._Caffeine_trial_2.png|left|thumb|100pxs|Trial 2]] | ||
Latest revision as of 10:11, 16 December 2022
Contents
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
Procedure:
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
- Vortex the yeast briefly before use.
- To the first well, add 50 ul of 0.1 M stock solution and 50 ul of yeast.
- Repeat the first step in the next well for 0.05 M solution, continuing to add each stock solution to the next wells.
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
Procedure
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.
