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