View source for UW-Stout/Voltage SP23


== Pilot Experiment ==

'''Materials'''
*Ice Cold Water
*Ice-Cold Electroporation Buffer (1M D-Sorbitol)
*0.1M Lithium Acetate
*Room Temperature Electroporation Buffer (1M D-Sorbitol)
*Wild Type Yeast Culture

'''Equipment''' 
*Electroporator 
*Electroporation Cuvettes (Pre-Chilled)
*Centrifuge 
*Incubator (30 Degrees Celsius) 
*T200 Micropipettor
*Microcentrifuge Tubes
*Biosafety Cabinet
*YPD Plates

'''Protocol'''
# Pipette 110 microliters of wild type yeast culture into microcentrifuge tube. 
# Wash the cell pellet twice with 200 microliters of ice-cold water at 4500 rpm for 5 min. Discard supernatant. 
# Wash the cell pellet once with 200 microliters of ice-cold electroporation buffer (1M D-sorbitol) at 4500 rpm for 5 min. Discard supernatant.
# Wash the cell pellet with 200 microliters 0.1 M Lithium Acetate at 4500 rpm for 5 min. Discard supernatant. 
# Wash the cell pellet once with 200 microliters of ice-cold electroporation buffer at 4500 rpm for 5 min. Discard supernatant. 
# Suspend the pellet in 200 microliters of electroporation buffer. (Keep cells on ice until electroporation).
# Insert 100 microliters of the mixture into pre-chilled electroporation cuvette. Keep cuvette on ice for 5 minutes before electroporation. 
# Electroporate the cells at wanted voltage. 
# Insert 100 microliters of YPD into cuvette and place 50 microliters of the mixture on YPD plates to be examined. Place 50 microliters of the non-electroporated mixture onto negative control plates.  

'''Data'''

[[Image:Pilot2All.jpg]]

'''Interpretation'''

Based on the results of the pilot experiment, we decided on 2.5kV electroporation. This voltage showed the greatest amount of cell stress/death. This was the highest voltage tested.

== Final Protocol (Knockout Experiment) ==

'''Materials'''
*Ice Cold Water
*Ice-Cold Electroporation Buffer (1M D-Sorbitol)
*0.1M Lithium Acetate
*Room Temperature Electroporation Buffer (1M D-Sorbitol)
*Knockout Yeast Strains 
**[[YMR144W]]
**[[YLR006C]]
**[[YKL162C]]

'''Equipment''' 
*Electroporator 
*Electroporation Cuvettes (Pre-Chilled)
*Centrifuge 
*Incubator (30 Degrees Celsius) 
*T200 Micropipettor
*Microcentrifuge Tubes
*Biosafety Cabinet
*YPD Plates

'''Protocol''' 
# Pipette 110 microliters of each knockout strain into microcentrifuge tubes. 
# Wash the cell pellets twice with 200 microliters of ice-cold water at 4500 rpm for 5 min. Discard supernatant. 
# Wash the cell pellets once with 200 microliters of ice-cold electroporation buffer (1M D-sorbitol) at 4500 rpm for 5 min. Discard supernatant.
# Wash the cell pellets with 200 microliters 0.1 M Lithium Acetate at 4500 rpm for 5 min. Discard supernatant. 
# Wash the cell pellets once with 200 microliters of ice-cold electroporation buffer at 4500 rpm for 5 min. Discard supernatant. 
# Suspend the pellets in 200 microliters of electroporation buffer. (Keep cells on ice until electroporation).
# Insert 100 microliters of each mixture into pre-chilled electroporation cuvettes. Keep cuvettes on ice for 5 minutes before electroporation. 
# Electroporate the cells at 2.5kV. 
# Insert 100 microliters of YPD into each cuvette and place 50 microliters of each mixture on YPD plates to be examined. Place 50 microliters of the non-electroporated mixture onto negative control plates.