Difference between revisions of "UW-Stout/Bases SP23"
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*D5 is the pH level 10 | *D5 is the pH level 10 | ||
*D6 is the pH level 10.5 | *D6 is the pH level 10.5 | ||
− | *D7 is the wild type control group no buffer added | + | *D7 is the wild type control group no buffer added |
− | + | [[Media:EMPilotTrial3-Bases.jpg]] | |
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='''Final Experiment'''= | ='''Final Experiment'''= |
Revision as of 12:19, 2 May 2023
Contents
Pilot Experiment
Introduction
The goal of the experiment is to find a pH level of ammonium hydroxide and ammonium chloride buffer that upsets our yeast but does not kill them. The pH levels range from 8 to 10.5 increasing in .5 increments and then recording the effects on the growth at each level.
Materials
- Ammonium Chloride
- Ammonium Hydroxide
- Corning COSTAR 96-well clear flat-bottom assay plate
- microcentrifuge tubes
- micropipette and corresponding tips
- p20
- p100
- p1000
- pH reader
- wild-type yeast in 2x synthesis complete media at an OD600 of 0.1-0.2
- 15 mL conical tubes (x6)
- 20 mL sterile water
Procedure
Make basic buffers using the ammonium chloride and ammonium hydroxide
- add small increments of the ammonium hydroxide into the ammonium chloride while using the pH reader until you reach pH level of 10.5
- pour buffer into a labeled conical tube
- repeat step 1 and 2 until you have made each pH level
- 8 8.5 9 9.5 10 10.5
- once buffers are made they can be stored at room temperature
this portion of the experiment is done in a sterile environment like a biosafety cabinet
MAKE SURE TO VORTEX YEAST CELLS BEFORE ADDING TO MICROCENTRIFUGE TUBES
- in a labeled microcentrifuge tube add
- 50 ul yeast culture
- 40 ul sterile water
- 10 ul of pH level 8 buffer
- repeat step above using each pH level
- for the control in a labeled microcentrifuge tube add
- 50 ul sterile water
- 50 ul yeast culture
- vortex each tube
- pipette 100ul of each concentration into a well
- make sure to record which pH level is in each well
- set up plate reader
- temperature 30 degrees Celsius
- mode kinetic
- wavelength 600 nm
- interval 5 minutes
- total run time 24 hours
- shake before read 30 seconds
- transfer assay plate into reader and read for 24 hours
Data Recording
Pilot Trial 3 Bases Graph
- D1 is the pH level 8
- D2 is the pH level 8.5
- D3 is the pH level 9
- D4 is the pH level 9.5
- D5 is the pH level 10
- D6 is the pH level 10.5
- D7 is the wild type control group no buffer added
Final Experiment
Introduction
Materials
- ammonium chloride and ammonium hydroxide buffer at pH level 8.5
- Corning COSTAR 96-well clear flat-bottom assay plate
- microcentrifuge tubes
- micropipette and corresponding tips
- p20
- p100
- p1000
- pH reader
- yeast strain 1 YMR144W
- yeast strain 2 SSK1
- yeast strain 3 YKL162C
- wild-type yeast BY4735
- 20 mL sterile water
Procedure
this portion of the experiment is done in a sterile environment like a biosafety cabinet
MAKE SURE TO VORTEX YEAST CELLS BEFORE ADDING TO MICROCENTRIFUGE TUBES
- in a labeled microcentrifuge tube add
- 50 ul yeast strain 1
- 40 ul sterile water
- 10 ul of pH level 8.5 buffer
- repeat step above with yeast strain 1 for a total of three samples
- vortex each tube
- pipette 100ul of each sample into a well
- make sure to record which yeast strain sample is in each well
- in a labeled microcentrifuge tube add
- 50 ul yeast strain 2
- 40 ul sterile water
- 10 ul of pH level 8.5 buffer
- repeat step above with yeast strain 2 for a total of three samples
- vortex each tube
- pipette 100ul of each sample into a well
- make sure to record which yeast strain sample is in each well
- in a labeled microcentrifuge tube add
- 50 ul yeast strain 3
- 40 ul sterile water
- 10 ul of pH level 8.5 buffer
- repeat step above with yeast strain 3 for a total of three samples
- vortex each tube
- pipette 100ul of each sample into a well
- make sure to record which yeast strain sample is in each well
- in a labeled microcentrifuge tube add
- 50 ul wild type yeast
- 40 ul sterile water
- 10 ul of pH level 8.5 buffer
- repeat step above with wild type yeast for a total of three samples
- vortex each tube
- pipette 100ul of each sample into a well
- make sure to record which yeast strain sample is in each well
- set up plate reader
- temperature 30 degrees Celsius
- mode kinetic
- wavelength 600 nm
- interval 5 minutes
- total run time 24 hours
- shake before read 30 seconds
- transfer assay plate into reader and read for 24 hours