Difference between revisions of "UW-Stout/Formamide SP22"

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*The 3% formamide used in the pilot experiment and represented as B- was chosen to be used on the transformed yeast cells
+
*The 3% formamide used in the pilot experiment and represented as B-5 (in the top-most picture) was chosen to be used on the transformed yeast cells
*This was deduced due to the position of B-  
+
*This was deduced due to the position of B-
*B- was able to grow, but didn't grow too much- simply put, it was in the middle
+
*B-5 was able to grow, but didn't grow too much- simply put, it was in the middle
  
 
==Knockout Yeast Cell Gene==
 
==Knockout Yeast Cell Gene==

Revision as of 17:16, 9 May 2022

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
  • Well plate
  • Wild yeast cells, 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. Use the 13th PCR tube to transfer the amount of formamide to each PCR tube as needed
  5. Pipet appropriate amount of formamide and sterile water into the labeled PCR tubes according to number
  6. In a sterile environment, pipet the liquid (50ul) from the PCR tube into the well cell as well as 50ul of the wild yeast cells
  7. Incubate assess tray at 30 degrees Celsius for 24 hours and record data

Results

pilot.jpg


  • The x-axis corresponds to the concentrations listed up above (ex: B1 corresponds to the 0.5%)
  • B-12 is an experimental error- since it has a concentration of 0% formamide anyway, it was left in the final graph


3formamide.jpg


  • The 3% formamide used in the pilot experiment and represented as B-5 (in the top-most picture) was chosen to be used on the transformed yeast cells
  • This was deduced due to the position of B-5
  • B-5 was able to grow, but didn't grow too much- simply put, it was in the middle

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. Grab neat formamide and sterile water
  2. Label PCR tubes 1-9
  3. Pipet approximately 18ul formamide into a PCR tube, can put excess in
  4. Pipet 3.0ul of formamide into each of the numbered PCR tubes
  5. Pipet 47.0ul of sterile water into each of the numbered PCR tubes
  6. In a sterile environment, pipet 50ul of liquid from the PCR tubes into well cells. Pipet 50ul of each knockout strain into the appropriate well cells
  7. Incubate assess tray at 30 degrees Celsius for 24 hours and record data

Results