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

Revision as of 18:40, 9 May 2022

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

Obtain the neat formamide (refrigerated) and sterile water
Obtain and label 12 PCR tubes 1-12
Obtain one more PCR tube to transfer 33ul of neat formamide inside, can put excess in
Using the 13th PCR tube, pipet appropriate amount of formamide and sterile water into the labeled PCR tubes according to number
In a sterile environment, pipet the liquid (50ul) from the PCR tube into the well cell
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
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
Transfer the assay plate to the reader and read for 24 hours

The x-axis corresponds to the concentrations listed up above (ex: B1 corresponds to the 0.5%)
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 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

Obtain neat formamide (refrigerated) and sterile water
Obtain and label PCR tubes 1-9
Pipet approximately 18ul formamide into a 10th PCR tube, can put excess in
Using the 10th PCR tube, pipet 3.0ul of formamide into each PCR tube (labeled 1-9)- this minimizes contamination
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
Incubate assess tray at 30 degrees Celsius for 24 hours and record data

Ran three trials, trial one through three is posted up above in picture form
The dark line in the middle of the graph 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

@@ Line 32: / Line 32: @@
 # Obtain and label 12 PCR tubes 1-12
 # Obtain one more PCR tube to transfer 33ul of neat formamide inside, can put excess 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 liquid (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
-# 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
 ===Results===