Difference between revisions of "UW-Stout/Hydroxychloroquine SP21"
(→Calibration Protocol) |
(→Calibration Protocol) |
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#Vortex the yeast culture briefly to resuspend the yeast cells. | #Vortex the yeast culture briefly to resuspend the yeast cells. | ||
#Prepare a stock solution of hydroxychloroquine 3000 ug/mL. | #Prepare a stock solution of hydroxychloroquine 3000 ug/mL. | ||
| − | #Set up 8 wells | + | #Set up 8 wells as follows: |
| + | #*Well 1: 3.3uL HCQ, 46.7uL sterile water (final concentration 100ug/mL) | ||
| + | #*Well 2: 10uL HCQ, 40uL sterile water (final concentration 300ug/mL) | ||
| + | #*Well 3: 16.7uL HCQ, 33.3uL sterile water (final concentration 500ug/mL) | ||
| + | #*Well 4: 23.3uL HCQ, 26.7uL sterile water (final concentration 700ug/mL) | ||
| + | #*Well 5: 30uL HCQ, 20uL sterile water (final concentration 900ug/mL) | ||
| + | #*Well 6: 36.7uL HCQ, 13.3uL sterile water (final concentration 1100ug/mL) | ||
| + | #*Well 7: 43.3uL HCQ, 6.7uL sterile water (final concentration 1300ug/mL) | ||
| + | #*Well 8: 50uL HCQ (final concentration 1500ug/mL) | ||
| + | #Add 50uL of yeast culture to each well. | ||
#Aliquot 50 uL of each stock and add 50 uL of yeast culture to each sample. | #Aliquot 50 uL of each stock and add 50 uL of yeast culture to each sample. | ||
#To measure cell growth over a period of 24 hours, set up the plate reader as follows: | #To measure cell growth over a period of 24 hours, set up the plate reader as follows: | ||
Revision as of 09:51, 27 April 2021
Materials
- Corning COSTAR 96-well clear flat-bottom assay plate
- Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2
- Hydroxychloroquine, 300 ug/uL solution in sterile water (as calculated in the calibration protocol)
- P20, P200 micropipettors
Equipment
Precautions
Use gloves and safety glasses for this experiment. Hydroxychloroquine is a potent chemical used to treat malaria and some autoimmune conditions. However, side effects include but are not limited to nausea, headache, dizziness, rash.
Calibration Protocol
- Vortex the yeast culture briefly to resuspend the yeast cells.
- Prepare a stock solution of hydroxychloroquine 3000 ug/mL.
- Set up 8 wells as follows:
- Well 1: 3.3uL HCQ, 46.7uL sterile water (final concentration 100ug/mL)
- Well 2: 10uL HCQ, 40uL sterile water (final concentration 300ug/mL)
- Well 3: 16.7uL HCQ, 33.3uL sterile water (final concentration 500ug/mL)
- Well 4: 23.3uL HCQ, 26.7uL sterile water (final concentration 700ug/mL)
- Well 5: 30uL HCQ, 20uL sterile water (final concentration 900ug/mL)
- Well 6: 36.7uL HCQ, 13.3uL sterile water (final concentration 1100ug/mL)
- Well 7: 43.3uL HCQ, 6.7uL sterile water (final concentration 1300ug/mL)
- Well 8: 50uL HCQ (final concentration 1500ug/mL)
- Add 50uL of yeast culture to each well.
- Aliquot 50 uL of each stock and add 50 uL of yeast culture to each sample.
- To measure cell growth over a period of 24 hours, set up the plate reader as follows:
- Temperature: 30°C
- Mode: Kinetic
- Wavelength: 600 nm
- Interval: 5 minutes
- Total run time: 24 hours
- Shake before reading: 30 seconds.
- Transfer the assay plate to the reader and read for 24 hours.
Protocol
- Vortex the yeast culture briefly to resuspend the yeast cells.
- Set up seven wells aliquoting 50uL of the stock hydroxychloroquine and 50uL of yeast culture. Use the seventh well for the wild-type yeast strain.
- The final concentration of hydroxychloroquine is 300ug/mL.
- To measure cell growth over a period of 24 hours, set up the plate reader as follows:
- Temperature: 30°C
- Mode: Kinetic
- Wavelength: 600 nm
- Interval: 5 minutes
- Total run time: 24 hours
- Shake before reading: 30 seconds
- Transfer the assay plate to the reader and read for 24 hours.
- Note: Repeat the experiment a few times to account for natural variation.
