View source for 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
*Well plate
*Wild yeast cells, 600ul
*Molecular Devices SpectraMax Plus 384 Microplate Reader
*Micropipet 100ul 
*Micropipet tips

===Formamide Concentrations===
# 0.5ul formamide + 49.5ul sterile water
# 1.0ul formamide + 49.0ul sterile water
# 1.5ul formamide + 48.5ul sterile water
# 2.0ul formamide + 48.0ul sterile water
# 2.5ul formamide + 47.5ul sterile water
# 3.0ul formamide + 47.0ul sterile water 
# 3.5ul formamide + 46.5ul sterile water
# 4.0ul formamide + 46.0ul sterile water 
# 4.5ul formamide + 45.5ul sterile water
# 5.0ul formamide + 45.0ul sterile water
# 5.5ul formamide + 44.5ul sterile water
# 0.0ul formamide + 50.0ul sterile water

===Procedure===
# 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
# Use the 13th PCR tube to transfer the amount of formamide to each PCR tube as needed 
# 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 as well as 50ul of the wild yeast cells
# Incubate assess tray at 30 degrees Celsius for 24 hours and record data

===Results===
[[File:pilot1.jpg|300px|]]


*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


[[File:3formamide.jpg|300px|]]


*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===
# 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

===Results===
[[File:trial11.jpg|300px|]]


[[File:trial22.jpg|300px|]]


[[File:trial3.jpg|300px|]]


*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