Difference between revisions of "UW Stout/DMSO FA19"

From SGD-Wiki
Jump to: navigation, search
(Created page with " == Preliminary Yeast Torture Protocol == '''Materials:''' *Wild Type Yeast *DMSO at 100% solution *Sterile H<sub>2</sub>O *96 well plate '''Equipment''' *P2 Micropipette *P...")
 
(→‎Preliminary Yeast Torture Protocol)
 
(2 intermediate revisions by the same user not shown)
Line 40: Line 40:
 
*20%= 30 &mu;liters of water, 20 &mu;liters of DMSO at 100%, 50 &mu;liters of yeast solution
 
*20%= 30 &mu;liters of water, 20 &mu;liters of DMSO at 100%, 50 &mu;liters of yeast solution
 
*50%= 50 &mu;liters of DMSO at 100%, and 50 &mu;liters of yeast solution
 
*50%= 50 &mu;liters of DMSO at 100%, and 50 &mu;liters of yeast solution
 +
 +
'''Results'''
 +
 +
[[Image:Calibration_Experiment_DMSO.png]]
 +
 +
The graph above shows the effects of different percent DMSO solutions on wild type yeast. Two percent DMSO solution had
 +
very little effect on the wild type yeast and five percent DMSO had too large an impact for the intended use.
 +
From this information it was decided to use a three percent solution for future experiments with the different knockout strains of yeast.
 +
 +
==Oxidative Stress on Yeast Through the Use of 3% DMSO Protocol==
 +
 +
'''Materials'''
 +
*Wild Type Yeast transformation BY4735
 +
*Yeast transformations with YAL061W knockout
 +
*Yeast transformation with YBL086C knockout
 +
*Yeast transformation with YBL081W knockout
 +
*Yeast transformation with YBR033W knockout
 +
*Yeast transformation with YBR138C knockout
 +
*Yeast transformation with YBR220C knockout
 +
*Yeast transformation with YHR048W knockout
 +
*Yeast transformation with YGL140C knockout
 +
*Yeast transformation with YJR061W knockout
 +
*Yeast transformation with YKR 005C knockout
 +
*DMSO at 100% solution
 +
*Sterile H<sub>2</sub>O
 +
*96 well plate
 +
 +
'''Equipment'''
 +
*P10 Micropipette
 +
*P200 Micropipette
 +
*Molecular Devices SpectraMax Plus 384 Microplate Reader
 +
 +
'''Protocol'''
 +
 +
#Label wells in accordance to yeast transformation type.
 +
#Add 50 &mu;liter of yeast transformation into the corresponding well on the 96 well plate
 +
#Add 47 &mu;liter of H2O into each well
 +
#Add 3 &mu;liter of 100%  DMSO into each
 +
#Incubate in Microplate reader for 6-24 hours at 30&deg;C
 +
#Collect and analyze data

Latest revision as of 07:17, 9 December 2019

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

Results

Calibration Experiment DMSO.png

The graph above shows the effects of different percent DMSO solutions on wild type yeast. Two percent DMSO solution had very little effect on the wild type yeast and five percent DMSO had too large an impact for the intended use. From this information it was decided to use a three percent solution for future experiments with the different knockout strains of yeast.

Oxidative Stress on Yeast Through the Use of 3% DMSO Protocol

Materials

  • Wild Type Yeast transformation BY4735
  • Yeast transformations with YAL061W knockout
  • Yeast transformation with YBL086C knockout
  • Yeast transformation with YBL081W knockout
  • Yeast transformation with YBR033W knockout
  • Yeast transformation with YBR138C knockout
  • Yeast transformation with YBR220C knockout
  • Yeast transformation with YHR048W knockout
  • Yeast transformation with YGL140C knockout
  • Yeast transformation with YJR061W knockout
  • Yeast transformation with YKR 005C knockout
  • DMSO at 100% solution
  • Sterile H2O
  • 96 well plate

Equipment

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

Protocol

  1. Label wells in accordance to yeast transformation type.
  2. Add 50 μliter of yeast transformation into the corresponding well on the 96 well plate
  3. Add 47 μliter of H2O into each well
  4. Add 3 μliter of 100% DMSO into each
  5. Incubate in Microplate reader for 6-24 hours at 30°C
  6. Collect and analyze data
Retrieved from "https://wiki.yeastgenome.org/index.php?title=UW_Stout/DMSO_FA19&oldid=401523"