Difference between revisions of "UW-Stout/Bases SP23"
| Line 1: | Line 1: | ||
| − | '''Introduction''' | + | =='''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. | 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''' | + | =='''Materials'''== |
*Ammonium Chloride | *Ammonium Chloride | ||
*Ammonium Hydroxide | *Ammonium Hydroxide | ||
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*20 mL sterile water | *20 mL sterile water | ||
| − | '''Procedure''' | + | =='''Procedure'''== |
Make basic buffers using the ammonium chloride and ammonium hydroxide | 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 | # add small increments of the ammonium hydroxide into the ammonium chloride while using the pH reader until you reach pH level of 10.5 | ||
| Line 42: | Line 42: | ||
* shake before read 30 seconds | * shake before read 30 seconds | ||
#transfer assay plate into reader and read for 24 hours | #transfer assay plate into reader and read for 24 hours | ||
| − | '''Data Recording''' | + | =='''Data Recording'''== |
Revision as of 06:12, 28 April 2023
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
- 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
