UW-Stout/Voltage SP23

Pilot Experiment

Materials

• Ice Cold Water
• Ice-Cold Electroporation Buffer (1M D-Sorbitol)
• 0.1 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 1. Pipette 110 microliters of wild type yeast culture into microcentrifuge tube. 2. Wash the cell pellet twice with 200 microliters of ice-cold water at 4500 rpm for 5 min. Discard supernatant. 3. Wash the cell pellet once with 200 microliters of ice-cold electroporation buffer (1M D-sorbitol) at 4500 rpm for 5 min. Discard supernatant. 4. Wash the cell pellet with 200 microliters 0.1 M Lithium Acetate at 4500 rpm for 5 min. Discard supernatant. 5. Wash the cell pellet once with 200 microliters of ice-cold electroporation buffer at 4500 rpm for 5 min. Discard supernatant. 6. Suspend the pellet in 200 microliters of electroporation buffer. (Keep cells on ice until electroporation). 7. Insert 100 microliters of the mixture into pre-chilled electroporation cuvette. Keep cuvette on ice for 5 minutes before electroporation. 8. Electroporate the cells at wanted voltage. 9. 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

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)

1. Pipette 110 microliters of each knockout strain into microcentrifuge tubes. 2. Wash the cell pellets twice with 200 microliters of ice-cold water at 4500 rpm for 5 min. Discard supernatant. 3. Wash the cell pellets once with 200 microliters of ice-cold electroporation buffer (1M D-sorbitol) at 4500 rpm for 5 min. Discard supernatant. 4. Wash the cell pellets with 200 microliters 0.1 M Lithium Acetate at 4500 rpm for 5 min. Discard supernatant. 5. Wash the cell pellets once with 200 microliters of ice-cold electroporation buffer at 4500 rpm for 5 min. Discard supernatant. 6. Suspend the pellets in 200 microliters of electroporation buffer. (Keep cells on ice until electroporation). 7. Insert 100 microliters of each mixture into pre-chilled electroporation cuvettes. Keep cuvettes on ice for 5 minutes before electroporation. 8. Electroporate the cells at 2.5kV. 9. 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.