UW Stout/Cycloheximide FA19

Protocol Finding Correct Dilution of Cycloheximide

Materials List

• Wild type yeast (control)
• Cycloheximide
  • .01 grams
• Sterile H2O
• 96 well plate
• Plate Micro Readers
  • 30 Degrees Celsius
• Our yeast cells

Equipment

• Micropipettes
• Incubator set to 30 degrees Celsius
• New Brunswick TC-7 Tissue Culture Roller Drum
• Molecular Devices SpectraMax Plus 384 Microplate Reader

Protocol

1. Make a .001 solution
2. Label all wells being used
3. Enter in 50 Microliters of wild type yeast into each well
4. Then enter in each dilution mixture 1 for each well

• Add 1 microliter of .001 solution Cycloheximide in 49 microliters H2O
• Add 5 microliters of solution .001 Cycloheximide in 45 microliters H2O
• Add 1 microliters of solution .01 Cycloheximide in 49 microliters H2O
• Add 5 microliters of solution .01 Cycloheximide in 45 microliters H2O
• Add 10 microliters of solution .01 Cycloheximide in 40 microliters H2O
• Add 5 microliters of solution 1 Cycloheximide in 45 microlitersH2O
• Add 10 microliters of solution 1 Cycloheximide in 40 microliters H2O
• Add 15 microliters of solution 1 Cycloheximide in 35 microliters H2O
• Add 25 microliters of solution 1 Cycloheximide in 25 microliters H2O
• Add 35 microliters of solution 1 Cycloheximide in 15 microliters H2O
• Add 50 microliters of solution 1 Cycloheximide in 0 microliters H2O
• Control is just 50 microliters water

1. Incubate in Microplate reader for 6-8hrs at 30 degrees C
2. Collect data of which yeast cells did not thrive, but did not die, and find the optimal percentage of diluted cycloheximide

The data we found was that well two worked the best for optimal stressed induced to the yeast cells. Add 5 microliters of solution .001 cycloheximide in 49 microliters sterile H20.