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

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Line 5: Line 5:
  
 
===Materials / Equipment===
 
===Materials / Equipment===
−
*Neat Formamide, 33ul
+
*Neat Formamide, '''33ul'''
−
*Sterile water, 567ul
+
*Sterile water, '''567ul'''
−
*0.2ml flat cap PCR tube, 13
+
*0.2ml flat cap PCR tube, '''13 tubes'''
−
*Well plate
+
*Corning COSTAR 96-well clear flat-bottom assay plate
−
*Wild yeast cells, 600ul
+
*Wild-type yeast in 2x synthetic complete media at an OD600 of 0.1-0.2, '''600ul'''
 
*Molecular Devices SpectraMax Plus 384 Microplate Reader
 
*Molecular Devices SpectraMax Plus 384 Microplate Reader
−
*Micropipet 100ul  
+
*Micropipet (100ul)
 +
*Micropipet (10ul)
 +
*Micropipet (2ul)
 
*Micropipet tips
 
*Micropipet tips
  
Line 31: Line 33:
 
# Obtain the neat formamide (refrigerated) and sterile water
 
# Obtain the neat formamide (refrigerated) and sterile water
 
# Obtain and label 12 PCR tubes 1-12
 
# Obtain and label 12 PCR tubes 1-12
−
# Obtain one more PCR tube to transfer 33ul of neat formamide inside, can put excess in
+
# Obtain one more PCR tube to transfer 33ul of neat formamide inside, can put excess formamide in
−
# Use the 13th PCR tube to transfer the amount of formamide to each PCR tube as needed
+
# Using the 13th PCR tube, pipet appropriate amount of formamide and sterile water into the labeled PCR tubes according to number
−
# Pipet appropriate amount of formamide and sterile water into the labeled PCR tubes according to number
+
# In a sterile environment, pipet the solution (50ul) from the PCR tube into the well cell  
−
# 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
+
# 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-water solution
−
# Incubate assess tray at 30 degrees Celsius for 24 hours and record data
+
# Set up the plate reader as follows:
 +
## Temperature: 30 degrees Celsius
 +
## Mode: kinetic
 +
## Wavelength: 600 nm
 +
## Interval: 5 minutes
 +
## Total run time: 24 hours
 +
## Shake before read: 30 seconds
 +
# Transfer the assay plate to the reader and read for 24 hours
 +
# Record data
  
 
===Results===
 
===Results===
 
[[File:pilot1.jpg|300px|]]
 
[[File:pilot1.jpg|300px|]]
  
−
 
+
* The x-axis is in time (min) and the y-axis is optical density at 600
−
*The x-axis corresponds to the concentrations listed up above (ex: B1 corresponds to the 0.5%)
+
*The x-axis corresponds to the concentrations listed up above under the title "formamide concentrations"
−
*B-12 is an experimental error- since it has a concentration of 0% formamide anyway, it was left in the final graph
+
*0% is an experimental error- since it has a concentration of 0% formamide anyway, it was left in the final graph
  
  
Line 48: Line 58:
  
  
−
*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
+
* The x-axis is in time (min) and the y-axis is optical density at 600
−
*This was deduced due to the position of B-5  
+
*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
−
*B-5 was able to grow, but didn't grow too much- simply put, it was in the middle
+
**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==
 
==Knockout Yeast Cell Gene==
  
 
===Materials / Equipment===
 
===Materials / Equipment===
−
*Neat formamide, 18ul
+
*Neat formamide, '''18ul'''
−
*Sterile water, 423ul
+
*Sterile water, '''423ul'''
−
*0.2ml flat cap PCR tube 10
+
*0.2ml flat cap PCR tube, '''10 tubes'''
 
*Well plate
 
*Well plate
 
*Knock out yeast cells
 
*Knock out yeast cells
 
*Molecular Devices SpectraMax Plus 384 Microplate Reader
 
*Molecular Devices SpectraMax Plus 384 Microplate Reader
−
*Micropipet 100ul
+
*Micropipet (100ul)
 +
*Micropipet (20ul)
 +
*Micropipet (2ul)
 
*Micropipet tips
 
*Micropipet tips
  
Line 67: Line 80:
 
# Obtain neat formamide (refrigerated) and sterile water
 
# Obtain neat formamide (refrigerated) and sterile water
 
# Obtain and label PCR tubes 1-9
 
# Obtain and label PCR tubes 1-9
−
# Pipet approximately 18ul formamide into a 10th PCR tube, can put excess in
+
# Pipet approximately 18ul formamide into a 10th PCR tube, can put excess formamide in
−
# Using the 10th PCR tube, pipet 3.0ul of formamide into each PCR tube (labeled 1-9)- this minimizes contamination
+
# Using the 10th PCR tube, pipet 3.0ul of formamide into each PCR tube (labeled 1-9)  
 
# Pipet 47.0ul of sterile water into each of the numbered PCR tubes
 
# Pipet 47.0ul of sterile water into each of the numbered PCR tubes
−
# 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
+
# In a sterile environment, pipet 50ul of formamide-water solution from the PCR tubes into well cells  
−
# Incubate assess tray at 30 degrees Celsius for 24 hours and record data
+
#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
 +
# Set up the plate reader as follows:
 +
## Temperature: 30 degrees Celsius
 +
## Mode: kinetic
 +
## Wavelength: 600 nm
 +
## Interval: 5 minutes
 +
## Total run time: 24 hours
 +
## Shake before read: 30 seconds
 +
# Transfer the assay plate to the reader and read for 24 hours
 +
# Record data
  
 
===Results===
 
===Results===
−
[[File:trial11.jpg|300px|]]
+
[[File:trial11.jpg|300px|]] [[File:trial22.jpg|300px|]] [[File:trial3.jpg|300px|]]
−
 
 
  
−
*Ran three trials, trial one is posted up above in picture form
+
* The x-axis is in time (min) and the y-axis is optical density at 600
−
*Computed doubling times from the average times of the three trials
+
*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

Latest revision as of 19:49, 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 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 (10ul)
  • Micropipet (2ul)
  • 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 formamide 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 solution (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-water solution
  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

pilot1.jpg

  • The x-axis is in time (min) and the y-axis is optical density at 600
  • 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


3formamide.jpg


  • The x-axis is in time (min) and the y-axis is optical density at 600
  • 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 tubes
  • Well plate
  • Knock out yeast cells
  • Molecular Devices SpectraMax Plus 384 Microplate Reader
  • Micropipet (100ul)
  • Micropipet (20ul)
  • Micropipet (2ul)
  • 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

trial11.jpg trial22.jpg trial3.jpg

  • The x-axis is in time (min) and the y-axis is optical density at 600
  • 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
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