UW-Stout/G-418

Materials

1. Corning COSTAR 96-well clear flat-bottom assay plate
2. Geneticin 100 mg/ml
3. Glycerol stocks of knockout strains
4. Water

Equipment

1. Micropipettes
2. 96-well assay plate

G-418 Calibration

1. In 96-well assay plate in well 1 add 90 ul of yeast BY4735 into the well plate with 10 ul of water for the control.
2. In the same well plate in well 2 add 90 ul of yeast BY4735 into the well plate with 10 ul of Geneticin (also known as G-418) with a concentration of 100 ul/ml
3. In the same well plate in well 3 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 50 ul/ml
4. In the same well plate in well 4 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 25 ul/ml
5. In the same well plate in well 5 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 12.5 ul/ml
6. In the same well plate in well 6 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 6.3 ul/ml
7. In the same well plate in well 7 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 3.1 ul/ml
8. In the same well plate in well 8 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 1.56 ul/ml
9. In the same well plate in well 9 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 0.78 ul/ml
10. In the same well plate in well 10 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 0.39 ul/ml
11. In the same well plate in well 11 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 0.2 ul/ml
12. In the same well plate in well 12 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 0.1 ul/ml

Calibration Data

From the graphs, we determined that 100 ul/ml was the best option to use for our knock-out experiment because it slowed down the growth of BY4735 without killing the yeast.

This graph shows the results of testing the absorbance of yeast with different amounts of G-418.

Knock-out Experiment

1. In 96-well assay plate in well 1 add 90 ul of yeast BY4735 into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
2. In same well assay plate in well 2 add 90 ul of yeast YEL035C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
3. In same well assay plate in well 3 add 90 ul of yeast YCL200C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
4. In same well assay plate in well 4 add 90 ul of yeast YFL064C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
5. In same well assay plate in well 5 add 90 ul of yeast YGL235W into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
6. In same well assay plate in well 6 add 90 ul of yeast YCR100C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
7. In same well assay plate in well 7 add 90 ul of yeast YJL043W into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
8. In same well assay plate in well 8 add 90 ul of yeast YPL068C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
9. In same well assay plate in well 9 add 90 ul of yeast YMR090W into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml