Difference between revisions of "UW-Stout/Prednisone FA19"
(→Protocol) |
|||
Line 11: | Line 11: | ||
*Molecular Devices SpectraMax Plus 384 Microplate Reader | *Molecular Devices SpectraMax Plus 384 Microplate Reader | ||
==Protocol== | ==Protocol== | ||
− | #Make stock solution by vortexing the following: | + | #Make the stock solution by vortexing the following: |
#*50 mg Prednisone | #*50 mg Prednisone | ||
#*10 ml ethanol | #*10 ml ethanol | ||
Line 17: | Line 17: | ||
#In each well, add 50 µl of wildtype yeast strain. | #In each well, add 50 µl of wildtype yeast strain. | ||
#Using the stock solution (P1), add 20 µl to the first well along with 30 µl of sterile H2O. | #Using the stock solution (P1), add 20 µl to the first well along with 30 µl of sterile H2O. | ||
− | #*This well should now have 100 µl of | + | #*This well should now have 100 µl total of solution. |
#Using the stock solution (P1), add 6 µl to the second well along with 44 µl of sterile H2O. | #Using the stock solution (P1), add 6 µl to the second well along with 44 µl of sterile H2O. | ||
− | #*This well should now have 100 µl of | + | #*This well should now have 100 µl total of solution. |
#Repeat this process for each set of wells, going down one dilution after each set of two wells. | #Repeat this process for each set of wells, going down one dilution after each set of two wells. | ||
#*Refer to the Well Concentrations chart. | #*Refer to the Well Concentrations chart. |
Revision as of 14:45, 10 December 2019
Materials
- Prednisone
- Absolute Ethanol
- Yeast Cultures
- Sterile Water
- Corning COSTAR 96-Well Clear Flat-Bottom Assay Plate
- Micropipettes
- Centrifuge Tubes
Equipment
- Incubator set to 30°C
- Molecular Devices SpectraMax Plus 384 Microplate Reader
Protocol
- Make the stock solution by vortexing the following:
- 50 mg Prednisone
- 10 ml ethanol
- Dilute down to needed percentages (below) using sterile water.
- In each well, add 50 µl of wildtype yeast strain.
- Using the stock solution (P1), add 20 µl to the first well along with 30 µl of sterile H2O.
- This well should now have 100 µl total of solution.
- Using the stock solution (P1), add 6 µl to the second well along with 44 µl of sterile H2O.
- This well should now have 100 µl total of solution.
- Repeat this process for each set of wells, going down one dilution after each set of two wells.
- Refer to the Well Concentrations chart.
- Dilute pure ethanol down to needed percentages (below).
- In 12 different wells, a new row, add 50 µl of wildtype yeast strain.
- Add the ethanol dilutions to the 12 new wells, using the same process as used above.
- This row will be used to compare the Prednisone treated yeasts’ growth rates.
- Shake all wells at 37°C for 24 hours.
- Plot data and select the Prednisone amount/concentration for the next test that shows results but doesn’t completely kill the yeast cultures.