UW-Stout/Formamide SP22

Wild Yeast Cell Pilot Procedure

Caution

Neat Formamide is harmful to the eyes, if swallowed, inhaled, or absorbed through the skin.

Materials / Equipment

• Neat Formamide, """33ul"""
• Sterile water, """567ul"""
• 0.2ml flat cap PCR tube, """13 tubes"""
• Corning COSTAR 96-well clear flat-bottom assay plate
• Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2, """600ul"""
• Molecular Devices SpectraMax Plus 384 Microplate Reader
• Micropipet 100ul
• Micropipet tips

Formamide Concentrations

1. 0.5ul formamide + 49.5ul sterile water
2. 1.0ul formamide + 49.0ul sterile water
3. 1.5ul formamide + 48.5ul sterile water
4. 2.0ul formamide + 48.0ul sterile water
5. 2.5ul formamide + 47.5ul sterile water
6. 3.0ul formamide + 47.0ul sterile water
7. 3.5ul formamide + 46.5ul sterile water
8. 4.0ul formamide + 46.0ul sterile water
9. 4.5ul formamide + 45.5ul sterile water
10. 5.0ul formamide + 45.0ul sterile water
11. 5.5ul formamide + 44.5ul sterile water
12. 0.0ul formamide + 50.0ul sterile water

Procedure

1. Obtain the neat formamide (refrigerated) and sterile water
2. Obtain and label 12 PCR tubes 1-12
3. Obtain one more PCR tube to transfer 33ul of neat formamide inside, can put excess in
4. Using the 13th PCR tube, pipet appropriate amount of formamide and sterile water into the labeled PCR tubes according to number
5. In a sterile environment, pipet the liquid (50ul) from the PCR tube into the well cell
6. Vortex the yeast culture briefly to resuspend the yeast cells, and then pipet 50ul of the wild yeast cells into each well in addition to the formamide
7. Set up the plate reader as follows:
   1. Temperature: 30 degrees Celsius
   2. Mode: kinetic
   3. Wavelength: 600 nm
   4. Interval: 5 minutes
   5. Total run time: 24 hours
   6. Shake before read: 30 seconds
8. Transfer the assay plate to the reader and read for 24 hours
9. Record data

Results

• The x-axis corresponds to the concentrations listed up above under the title "formamide concentrations"
• 0% is an experimental error- since it has a concentration of 0% formamide anyway, it was left in the final graph

• The 3% formamide used in the pilot experiment and represented as the light blue color (in the top-most picture) was chosen to be used on the transformed yeast cells
  • This was deduced due to the position of the yeast cells under 3% formamide stress
  • Under 3% formamide, the yeast cells were able to grow, but didn't grow too much- simply put, the 3% line was right in the middle of the graph

Knockout Yeast Cell Gene

Materials / Equipment

• Neat formamide, 18ul
• Sterile water, 423ul
• 0.2ml flat cap PCR tube 10
• Well plate
• Knock out yeast cells
• Molecular Devices SpectraMax Plus 384 Microplate Reader
• Micropipet 100ul
• Micropipet tips

Procedure

1. Obtain neat formamide (refrigerated) and sterile water
2. Obtain and label PCR tubes 1-9
3. Pipet approximately 18ul formamide into a 10th PCR tube, can put excess formamide in
4. Using the 10th PCR tube, pipet 3.0ul of formamide into each PCR tube (labeled 1-9)
5. Pipet 47.0ul of sterile water into each of the numbered PCR tubes
6. In a sterile environment, pipet 50ul of formamide-water solution from the PCR tubes into well cells
7. Vortex each yeast culture strain briefly to resuspend the cells, and then pipet 50ul of each knockout strain into the appropriate well cells in addition to the solution
8. Set up the plate reader as follows:
   1. Temperature: 30 degrees Celsius
   2. Mode: kinetic
   3. Wavelength: 600 nm
   4. Interval: 5 minutes
   5. Total run time: 24 hours
   6. Shake before read: 30 seconds
9. Transfer the assay plate to the reader and read for 24 hours
10. Record data

Results

• Ran three trials, trial one through three is posted up above in picture form as a line graph
• The dark line in the middle of trail 2 and 3 is a scanning error from the Molecular Devices SpectraMax Plus 384 Microplate Reader
• Computed doubling times from the average times of the three trials for further analyzation