Difference between revisions of "UW-Stout/G418 FA21"
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5. Observe the the growth curves. | 5. Observe the the growth curves. | ||
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'''Data''' | '''Data''' | ||
(Image will be added, can not figure out how to do it) | (Image will be added, can not figure out how to do it) | ||
Revision as of 22:03, 14 December 2021
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.
(Image will be added, can not figure out how to do it)
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)
