Difference between revisions of "UW-Stout/Bases SP23"
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# pour buffer into a labeled conical tube | # pour buffer into a labeled conical tube | ||
# repeat step 1 and 2 until you have made each pH level | # repeat step 1 and 2 until you have made each pH level | ||
− | *8 8.5 9 9.5 10 10.5 | + | #:*8 8.5 9 9.5 10 10.5 |
− | *once buffers are made they can be stored at room temperature | + | #:*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''' | ''' 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 | MAKE SURE TO VORTEX YEAST CELLS BEFORE ADDING TO MICROCENTRIFUGE TUBES | ||
+ | |||
# in a labeled microcentrifuge tube add | # in a labeled microcentrifuge tube add | ||
− | * 50 ul yeast culture | + | #:* 50 ul yeast culture |
− | * 40 ul sterile water | + | #:* 40 ul sterile water |
− | * 10 ul of pH level 8 buffer | + | #:* 10 ul of pH level 8 buffer |
# repeat step above using each pH level | # repeat step above using each pH level | ||
# vortex each tube | # vortex each tube | ||
# pipette 100ul of each concentration into a well | # pipette 100ul of each concentration into a well | ||
− | *make sure to record which pH level is in each well | + | #:*make sure to record which pH level is in each well |
#set up plate reader | #set up plate reader | ||
− | *temperature 30 degrees Celsius | + | #:*temperature 30 degrees Celsius |
− | *mode kinetic | + | #:*mode kinetic |
− | *wavelength 600 nm | + | #:*wavelength 600 nm |
− | *interval 5 minutes | + | #:*interval 5 minutes |
− | * total run time 24 hours | + | #:* total run time 24 hours |
− | * 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 | ||
Line 52: | Line 54: | ||
=='''Materials'''== | =='''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'''== | =='''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 |
Revision as of 12:08, 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
- 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
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