Difference between revisions of "UW-Stout/G418 SP23"
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===G418 Genticin=== | ===G418 Genticin=== | ||
− | In the spring of 2023, | + | In the spring of 2023, at the |
− | [https://uwstout.edu University of Wisconsin -- Stout] | + | [https://uwstout.edu University of Wisconsin -- Stout], the Intro Cell and Molecular Biology class |
− | [[UW-Stout/Knockout_Protocol| | + | [[UW-Stout/Knockout_Protocol|knocked out]] three [https://yeastmine.yeastgenome.org/yeastmine/bagDetails.do?scope=all&bagName=Uncharacterized_ORFs genes of unknown function] in a |
[https://www.atcc.org/en/Products/All/200897.aspx BY4735 strain] | [https://www.atcc.org/en/Products/All/200897.aspx BY4735 strain] | ||
− | background | + | background. This page is specifically about screening these knockout strains for phenotypes using G418 Genticin. |
'''''Materials:''''' | '''''Materials:''''' | ||
− | #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 |
− | # | + | #Tubes with each modified yeast strain ([[YMR144W]], [[SSK1]], and [[YKL162C]]) |
− | #100µL of 100mg/mL G418 geneticin (100 mM solution in water) | + | #100µL of 100mg/mL '''G418 geneticin''' (100 mM solution in water) |
− | #2mL sterile H2O3 | + | #2mL '''sterile H2O3''' |
#Petri dishes | #Petri dishes | ||
#Corning COSTAR 96-well clear flat-bottom assay plate | #Corning COSTAR 96-well clear flat-bottom assay plate | ||
Line 19: | Line 19: | ||
'''''Procedure:''''' | '''''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) | + | #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) | + | #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) | #''50µL of each'' (Logarithmic concentrations for pilot experiment) | ||
− | ##100µg/mL concentration (50µL of 100µg/mL G418 geneticin diluted solution 1) | + | ##'''100µg/mL''' concentration (50µL of 100µg/mL '''G418 geneticin''' ''diluted solution 1'') |
− | ##50µg/mL concentration (25µL of diluted solution 1 + 25 µL water) | + | ##'''50µg/mL''' concentration (25µL of ''diluted solution 1'' + 25 µL sterile water) |
− | ##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 sterile water) |
− | ##12µg/mL concentration (6µL of diluted solution 1 + 44 µL water) | + | ##'''12µg/mL''' concentration (6µL of ''diluted solution 1'' + 44 µL sterile water) |
− | ##6µg/mL concentration (3µL of diluted solution 1 + 47 µL water) | + | ##'''6µg/mL''' concentration (3µL of ''diluted solution 1'' + 47 µL sterile water) |
− | ##3µg/mL concentration (15µL of diluted solution 2 + 35µL water) | + | ##'''3µg/mL''' concentration (15µL of ''diluted solution 2'' + 35µL sterile water) |
− | ##2µg/mL concentration (10µL of diluted solution 2 + 40 µL water) | + | ##'''2µg/mL''' concentration (10µL of ''diluted solution 2'' + 40 µL sterile water) |
− | ##1µg/mL concentration (5µL of diluted solution 2 + 45 µL water) | + | ##'''1µg/mL''' concentration (5µL of ''diluted solution 2'' + 45 µL sterile water) |
− | ##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 sterile water) |
− | ##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 sterile water) |
− | ##0µg/mL concentration (50µL of sterile water) for control | + | ##'''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 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. | + | #Pipette 50µL of ''wild type yeast'' into each of the 10 micro wells in the plate. |
#Wait, then measure yeast growth. | #Wait, then measure yeast growth. | ||
#Vortex the yeast culture briefly to resuspend the yeast cells. | #Vortex the yeast culture briefly to resuspend the yeast cells. | ||
Line 45: | Line 45: | ||
##Shake before read: 30 seconds | ##Shake before read: 30 seconds | ||
#Transfer the assay plate to the reader and read for 24 hours. | #Transfer the assay plate to the reader and read for 24 hours. | ||
+ | |||
+ | '''''Results:''''' | ||
+ | |||
+ | the data collected from the experiment indicated that strain 2&3 were more impacted by geneticin with their respective gene knockout than strain 1 and wild type. |
Latest revision as of 14:46, 5 May 2023
G418 Genticin
In the spring of 2023, at the University of Wisconsin -- Stout, the Intro Cell and Molecular Biology class knocked out three genes of unknown function in a BY4735 strain background. This page is specifically about screening these knockout strains for phenotypes using G418 Genticin.
Materials:
- Wild type yeast tube in 2x synthetic complete media at an OD600 of 0.1-0.2
- Tubes with each modified yeast strain (YMR144W, SSK1, and YKL162C)
- 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 sterile water)
- 25µg/mL concentration (12.5µL of diluted solution 1 + 37.5 µL sterile water)
- 12µg/mL concentration (6µL of diluted solution 1 + 44 µL sterile water)
- 6µg/mL concentration (3µL of diluted solution 1 + 47 µL sterile water)
- 3µg/mL concentration (15µL of diluted solution 2 + 35µL sterile water)
- 2µg/mL concentration (10µL of diluted solution 2 + 40 µL sterile water)
- 1µg/mL concentration (5µL of diluted solution 2 + 45 µL sterile water)
- 0.5µg/mL concentration (2.5µL of diluted solution 2 + 47.5 µL sterile water)
- 0.25µg/mL concentration (1.25µL of diluted solution 2 + 48.75 µL sterile 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.
Results:
the data collected from the experiment indicated that strain 2&3 were more impacted by geneticin with their respective gene knockout than strain 1 and wild type.