UW-Stout/G418 SP23

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:

1. Wild type yeast tube in 2x synthetic complete media at an OD600 of 0.1-0.2
2. Tubes with each modified yeast strain (YMR144W, SSK1, and YKL162C)
3. 100µL of 100mg/mL G418 geneticin (100 mM solution in water)
4. 2mL sterile H2O3
5. Petri dishes
6. Corning COSTAR 96-well clear flat-bottom assay plate
7. Molecular Devices SpectraMax Plus 384 Microplate Reader

Procedure:

1. 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)
2. 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)
3. 50µL of each (Logarithmic concentrations for pilot experiment)
   1. 100µg/mL concentration (50µL of 100µg/mL G418 geneticin diluted solution 1)
   2. 50µg/mL concentration (25µL of diluted solution 1 + 25 µL sterile water)
   3. 25µg/mL concentration (12.5µL of diluted solution 1 + 37.5 µL sterile water)
   4. 12µg/mL concentration (6µL of diluted solution 1 + 44 µL sterile water)
   5. 6µg/mL concentration (3µL of diluted solution 1 + 47 µL sterile water)
   6. 3µg/mL concentration (15µL of diluted solution 2 + 35µL sterile water)
   7. 2µg/mL concentration (10µL of diluted solution 2 + 40 µL sterile water)
   8. 1µg/mL concentration (5µL of diluted solution 2 + 45 µL sterile water)
   9. 0.5µg/mL concentration (2.5µL of diluted solution 2 + 47.5 µL sterile water)
   10. 0.25µg/mL concentration (1.25µL of diluted solution 2 + 48.75 µL sterile water)
   11. 0µg/mL concentration (50µL of sterile water) for control
4. 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.
5. Pipette 50µL of wild type yeast into each of the 10 micro wells in the plate.
6. Wait, then measure yeast growth.
7. Vortex the yeast culture briefly to resuspend the yeast cells.
8. Set up the plate reader as follows:
   1. Temperature: 30°C
   2. Mode: Kinetic
   3. Wavelength: 600 nm
   4. Interval: 5 minutes
   5. Total run time: 24 hours
   6. Shake before read: 30 seconds
9. 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.

Gene:

the gene of focus in this protocol is the YMR144W gene. The gene has no known outcome on phenotype for yeast, through the experiment this was further proven. the use of geneticin on the yeast strain without YMR144W had a similar effect on wild type yeast. In looking into the data for the light capture, wild type yeast and the YMR144W knockout strain had similar growth at the end of the time period given for growth, as seen in figure 1.