UW-Stout/G-418
Contents
Materials
- Corning COSTAR 96-well clear flat-bottom assay plate
- Geneticin 100 mg/ml
- Glycerol stocks of knockout strains
- Water
Strains of Yeast
BY4735 (wild type), YEL035C, YCL002C, YFL064C, YGL235W, YCR100C, YJL043W, YPL068C, and YMR090W
Equipment
- Micropipettes
G-418 Calibration
- 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.
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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.
Knock-out Experiment
- 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
- 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
- In same well assay plate in well 3 add 90 ul of yeast YCL002C into the well plate with 10 ul of G-418 with a concentration of 100 ul/ml
- 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
- 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
- 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
- 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
- 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
- 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