UW Stout/DMSO FA19

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Preliminary Yeast Torture Protocol

Materials:

  • Wild Type Yeast
  • DMSO at 100% solution
  • Sterile H2O
  • 96 well plate

Equipment

  • P2 Micropipette
  • P10 Micropipette
  • P200 Micropipette
  • Molecular Devices SpectraMax Plus 384 Microplate Reader

Protocol

  1. Label wells in accordance to percent DMSO dilution
  2. Add 50 μliters of wild type yeast into the wells on the 96 well plate
  3. Add 49 μliters of sterile H2O into well labelled for 1% DMSO
  4. Add 1 μliters of DMSO at 100% into well labelled 1% DMSO
  5. Follow steps 2-4 for 0%, 0.1%, 0.2%, 0.5%, 2%, 5%, 10%, 20%, 50% DMSO desired
  6. Incubate in microplate reader for 6-12 hours at 30°C
  7. Collect and analyze data to determine optimal DMSO% for stressing yeast

Calculations

  • C1V1=C2V2
  • Make 50% solution of DMSO
  • Make 10% solution of DMSO
  • 100 μliters=V2
  • 0%= 50 μliters of water, and 50 μliters of yeast solution
  • 0.1% = 49 μliters of water, 1 μliters of DMSO at 10%, and 50 μliters of yeast solution
  • 0.2%= 48 μliters of water, 2 μliters of DMSO at 10%, and 50 μliters of yeast solution
  • 0.5%=49 μliters of water, 1 μliters of DMSO at 50%, and 50 μliters of yeast solution
  • 1%= 49 μliters of water, 1 μliter of DMSO at 100%, 50 μliters of yeast solution
  • 2%= 48 μliters of water, 2 μliters of DMSO at 100%, and 50 μliters of yeast solution
  • 5%= 45 μliters of water, 5 μliters of DMSO at 100%, and 50 μliters of yeast solution
  • 10% = 40 μliters of water, 10 μliters of DMSO at 100%, 50 μliters of yeast solution
  • 20%= 30 μliters of water, 20 μliters of DMSO at 100%, 50 μliters of yeast solution
  • 50%= 50 μliters of DMSO at 100%, and 50 μliters of yeast solution