Difference between revisions of "UW-Stout/Prednisone FA19"

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(Protocol)
(Protocol)
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#*50 mg Prednisone
 
#*50 mg Prednisone
 
#*10 ml ethanol
 
#*10 ml ethanol
#Dilute down to needed percentages ([[table]]) using sterile water.
+
#Dilute down to needed percentages (below) using sterile water.
 
#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 water.
 
#Using the stock solution (P1), add 20 µl to the first well along with 30 µl of sterile water.
Line 21: Line 21:
 
#*This well should now have 100 µl total of solution.
 
#*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]] table.
+
#*Refer to the Well Concentrations table (below).
#Dilute pure ethanol down to needed percentages ([[table]]).
+
#Dilute pure ethanol down to needed percentages.
 
#In 12 different wells, a new row, add 50 µl of wildtype yeast strain.
 
#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.
 
#Add the ethanol dilutions to the 12 new wells, using the same process as used above.

Revision as of 07:22, 12 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

  1. Make the stock solution by vortexing the following:
    • 50 mg Prednisone
    • 10 ml ethanol
  2. Dilute down to needed percentages (below) using sterile water.
  3. In each well, add 50 µl of wildtype yeast strain.
  4. Using the stock solution (P1), add 20 µl to the first well along with 30 µl of sterile water.
    • This well should now have 100 µl total of solution.
  5. Using the stock solution (P1), add 6 µl to the second well along with 44 µl of sterile water.
    • This well should now have 100 µl total of solution.
  6. Repeat this process for each set of wells, going down one dilution after each set of two wells.
    • Refer to the Well Concentrations table (below).
  7. Dilute pure ethanol down to needed percentages.
  8. In 12 different wells, a new row, add 50 µl of wildtype yeast strain.
  9. 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.
  10. Shake all wells at 37°C for 24 hours.
  11. Plot data and select the Prednisone amount/concentration for the next test that shows results but doesn’t completely kill the yeast cultures.