UW-Stout/G418 FA21

Calibration Protocol

Materials

Safety Gloves

Safety Glasses

Well Plate

Molecular Devices SpectraMax Plus 384 Microplate Reader

Fume Hood

Sterile Water

G418

Pipettes

Wild Type Yeast

5 Knockout Yeast

Protocol

1. Obtain 10 different concentrations of the G418 antibiotic and sterile water. Make sure to have 1 extra concentration of sterile water as a control growth curve of the wild type yeast.

2. Set up 11 wells shown in the "Well Components".

3. Put the well plate to the reader and read for 24 hours.

4. Observe the the growth curves.

5. Determine which concentration should be used on the knockout yeast.

Well Components

Well 1:

-50 ul wt yeast

-50 ul G418

Well 2:

-50 ul wt yeast

-16.7 ul G418

-33.3 ul water

Well 3:

-50 ul wt yeast

-5.6 ul G418

-44.5 ul water

Well 4:

-50 ul wt yeast

-1.9 ul G418

-48.2 ul water

Well 5:

-50 ul wt yeast

-0.63 ul G418

-49.4 ul water

Well 6:

-50 ul wt yeast

-0.2 ul G418

-49.8 ul water

Well 7:

-50 ul wt yeast

-0.07 ul G418

-49.93 ul water

Well 8:

-50 ul wt yeast

-0.02 ul G418

-49.98 ul water

Well 9:

-50 ul wt yeast

-0.0075 ul G418

-49.9925 ul water

Well 10:

-50 ul wt yeast

-0.0025 ul G418

-49.9975 ul water

Well 11:

-50 ul wt yeast

-50 ul water

Data

Based on the data from the Molecular Devices SpectraMax Plus 384 Microplate Reader, we decided to use the concentration from well 8 (E8).

KO Gene Protocol

Materials

Safety Gloves

Safety Glasses

Well Plate

Molecular Devices SpectraMax Plus 384 Microplate Reader

Fume Hood

Sterile Water

G418

Pipettes

Wild Type Yeast

5 Knockout Yeast

Protocol

1. Obtain the concentration of the G418 antibiotic and sterile water from the calibration protocol.

2. Set up 12 wells.

3. In the well plates there will be 2 of the same components. (There are 5 KO yeasts and 1 wild type)

4. Put the well plate to the reader and read for 24 hours.

5. Observe the the growth curves.

Data

(Image will be added, can not figure out how to do it)