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

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

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