Difference between revisions of "UW-Stout/Hydrogen Peroxide FA22"

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#Create dilutions from the stock hydrogen peroxide
 
#Create dilutions from the stock hydrogen peroxide
 
#Set up 12 wells in the assay plate according to the following table: [[Image:Pilot Setup.jpg|chart]]
 
#Set up 12 wells in the assay plate according to the following table: [[Image:Pilot Setup.jpg|chart]]
 +
#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 read: 30 seconds
 +
#Transfer the assay plate to the reader and read for 24 hours
 +
  
 
==Knockout Experiment==
 
==Knockout Experiment==

Revision as of 13:11, 7 December 2022

Introduction

We are using hydrogen peroxide to displaying varying growth of yeast cells. In this experiment, we tested many different concentrations then many different yeast cells with different genes knocked out.


Materials

  • 5000µM hydrogen peroxide solution
    • 3% hydrogen peroxide
    • Sterile water for dilutions
  • Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2
  • Yeast with different knocked out genes (except wild type, the gene listed is the one knocked out)
    • BY4735 (wild type)
    • YJL133C-A
    • YER186C
    • YER076C
    • YHR033W
    • YCR095C


Equipment

  • Molecular Devices SpectraMax Plus 384 Microplate Reader
  • Assay plate
  • Micropettes


Pilot Experiment

Protocol

  1. Vortex the BY4735 yeast culture briefly to resuspend the yeast cells.
  2. Create dilutions from the stock hydrogen peroxide
  3. Set up 12 wells in the assay plate according to the following table: chart
  4. 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 read: 30 seconds
  1. Transfer the assay plate to the reader and read for 24 hours


Knockout Experiment