Difference between revisions of "UW-Stout/G418 SP23"
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#Wild type yeast tube in 2x synthetic complete media at an OD600 of 0.1-0.2 | #Wild type yeast tube in 2x synthetic complete media at an OD600 of 0.1-0.2 | ||
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#Tube with my yeast strain | #Tube with my yeast strain | ||
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#100µL of 100mg/mL G418 geneticin (100 mM solution in water) | #100µL of 100mg/mL G418 geneticin (100 mM solution in water) | ||
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#2mL sterile H2O3 | #2mL sterile H2O3 | ||
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#Petri dishes | #Petri dishes | ||
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#Corning COSTAR 96-well clear flat-bottom assay plate | #Corning COSTAR 96-well clear flat-bottom assay plate | ||
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#Molecular Devices SpectraMax Plus 384 Microplate Reader | #Molecular Devices SpectraMax Plus 384 Microplate Reader | ||
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#Dilute G418 geneticin from 100mg/mL to 100µg/mL, making a total of 1mL (1000µL) of diluted solution 1 (with 100 µL of G418 geneticin and 900 µL of sterile water) | #Dilute G418 geneticin from 100mg/mL to 100µg/mL, making a total of 1mL (1000µL) of diluted solution 1 (with 100 µL of G418 geneticin and 900 µL of sterile water) | ||
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#Make second dilution of G418 geneticin from 100µg/mL to 10µg/mL, making 0.1mL (100µL) of diluted solution 2 (10 µL of first diluted solution + 90 µL of sterile water) | #Make second dilution of G418 geneticin from 100µg/mL to 10µg/mL, making 0.1mL (100µL) of diluted solution 2 (10 µL of first diluted solution + 90 µL of sterile water) | ||
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#''50µL of each'' (Logarithmic concentrations for pilot experiment) | #''50µL of each'' (Logarithmic concentrations for pilot experiment) | ||
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##100µg/mL concentration (50µL of 100µg/mL G418 geneticin diluted solution 1) | ##100µg/mL concentration (50µL of 100µg/mL G418 geneticin diluted solution 1) | ||
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##50µg/mL concentration (25µL of diluted solution 1 + 25 µL water) | ##50µg/mL concentration (25µL of diluted solution 1 + 25 µL water) | ||
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##25µg/mL concentration (12.5µL of diluted solution 1 + 37.5 µL water) | ##25µg/mL concentration (12.5µL of diluted solution 1 + 37.5 µL water) | ||
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##12µg/mL concentration (6µL of diluted solution 1 + 44 µL water) | ##12µg/mL concentration (6µL of diluted solution 1 + 44 µL water) | ||
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##6µg/mL concentration (3µL of diluted solution 1 + 47 µL water) | ##6µg/mL concentration (3µL of diluted solution 1 + 47 µL water) | ||
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##3µg/mL concentration (15µL of diluted solution 2 + 35µL water) | ##3µg/mL concentration (15µL of diluted solution 2 + 35µL water) | ||
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##2µg/mL concentration (10µL of diluted solution 2 + 40 µL water) | ##2µg/mL concentration (10µL of diluted solution 2 + 40 µL water) | ||
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##1µg/mL concentration (5µL of diluted solution 2 + 45 µL water) | ##1µg/mL concentration (5µL of diluted solution 2 + 45 µL water) | ||
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##0.5µg/mL concentration (2.5µL of diluted solution 2 + 47.5 µL water) | ##0.5µg/mL concentration (2.5µL of diluted solution 2 + 47.5 µL water) | ||
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##0.25µg/mL concentration (1.25µL of diluted solution 2 + 48.75 µL water) | ##0.25µg/mL concentration (1.25µL of diluted solution 2 + 48.75 µL water) | ||
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##0µg/mL concentration (50µL of sterile water) for control | ##0µg/mL concentration (50µL of sterile water) for control | ||
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#Pipette entire 50µL amount of ''each diluted G418 geneticin solution'' into each of the 10 micro wells to find out which concentration we will use for the final experiment. | #Pipette entire 50µL amount of ''each diluted G418 geneticin solution'' into each of the 10 micro wells to find out which concentration we will use for the final experiment. | ||
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#Pipette 50µL of wild type yeast into each of the 10 micro wells in the plate. | #Pipette 50µL of wild type yeast into each of the 10 micro wells in the plate. | ||
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#Wait, then measure yeast growth. | #Wait, then measure yeast growth. | ||
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#Vortex the yeast culture briefly to resuspend the yeast cells. | #Vortex the yeast culture briefly to resuspend the yeast cells. | ||
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#Set up the plate reader as follows: | #Set up the plate reader as follows: | ||
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##Temperature: 30°C | ##Temperature: 30°C | ||
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##Mode: Kinetic | ##Mode: Kinetic | ||
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##Wavelength: 600 nm | ##Wavelength: 600 nm | ||
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##Interval: 5 minutes | ##Interval: 5 minutes | ||
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##Total run time: 24 hours | ##Total run time: 24 hours | ||
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##Shake before read: 30 seconds | ##Shake before read: 30 seconds | ||
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#Transfer the assay plate to the reader and read for 24 hours. | #Transfer the assay plate to the reader and read for 24 hours. |
Revision as of 12:12, 20 April 2023
G418 Genticin
In the spring of 2023, one group in the Intro Cell and Molecular Biology class at the University of Wisconsin -- Stout began knocking out genes of unknown function in a BY4735 strain background, then screening those knockout strains for phenotypes. This page is specifically about the G418 Genticin experiment.
Materials:
- Wild type yeast tube in 2x synthetic complete media at an OD600 of 0.1-0.2
- Tube with my yeast strain
- 100µL of 100mg/mL G418 geneticin (100 mM solution in water)
- 2mL sterile H2O3
- Petri dishes
- Corning COSTAR 96-well clear flat-bottom assay plate
- Molecular Devices SpectraMax Plus 384 Microplate Reader
Procedure:
- Dilute G418 geneticin from 100mg/mL to 100µg/mL, making a total of 1mL (1000µL) of diluted solution 1 (with 100 µL of G418 geneticin and 900 µL of sterile water)
- Make second dilution of G418 geneticin from 100µg/mL to 10µg/mL, making 0.1mL (100µL) of diluted solution 2 (10 µL of first diluted solution + 90 µL of sterile water)
- 50µL of each (Logarithmic concentrations for pilot experiment)
- 100µg/mL concentration (50µL of 100µg/mL G418 geneticin diluted solution 1)
- 50µg/mL concentration (25µL of diluted solution 1 + 25 µL water)
- 25µg/mL concentration (12.5µL of diluted solution 1 + 37.5 µL water)
- 12µg/mL concentration (6µL of diluted solution 1 + 44 µL water)
- 6µg/mL concentration (3µL of diluted solution 1 + 47 µL water)
- 3µg/mL concentration (15µL of diluted solution 2 + 35µL water)
- 2µg/mL concentration (10µL of diluted solution 2 + 40 µL water)
- 1µg/mL concentration (5µL of diluted solution 2 + 45 µL water)
- 0.5µg/mL concentration (2.5µL of diluted solution 2 + 47.5 µL water)
- 0.25µg/mL concentration (1.25µL of diluted solution 2 + 48.75 µL water)
- 0µg/mL concentration (50µL of sterile water) for control
- Pipette entire 50µL amount of each diluted G418 geneticin solution into each of the 10 micro wells to find out which concentration we will use for the final experiment.
- Pipette 50µL of wild type yeast into each of the 10 micro wells in the plate.
- Wait, then measure yeast growth.
- Vortex the yeast culture briefly to resuspend the yeast cells.
- 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.