High-Efficiency Yeast Electroporation

Streak out yeast strains to be transformed onto YPD agar and grow at 30°C for 2 days.

For each transformation, inoculate cells into 4 separate tubes containing 5 ml of YPD, and grow to saturation overnight at 30°C with 270 rpm orbital shaking.

Pool the individual 5-ml overnight cultures into a sterile 50-ml conical tube and vortex to mix.

Measure the OD₆₀₀of the saturated cells.

• Generally need to make a 1:20 or 1:50 dilution to be within the linear range of the spectrophotometer.

In a 500-ml flask, dilute cultures to be transformed to a starting OD₆₀₀ of 2 in 100 ml of 2x YPD.

Example: If the overnight culture has an OD₆₀₀ of 10, use the solution dilution equation (C₁V₁ = C₂V₂) to solve for the amount of cells to add:

• C₁ = 2 OD₆₀₀ units (target OD₆₀₀), V₁ = 100 ml (target volume), C₂ = 10 OD₆₀₀ units (saturated OD₆₀₀), V₂ = ? (what we are solving for)
• Rearrange the equation: V₂ = C₁V₁ / C₂
• V2 = (2 OD₆₀₀ units x 100 ml) / 10 OD₆₀₀ units = 20-ml saturated cells
• For 100 ml total, add 20-ml saturated cells to 80-ml 2x YPD.

Grow the 100-ml inoculated cultures at 30°C with 270 rpm orbital shaking for 4 hours.

Place sterile water on ice and store at 4°C until ready to use.

• 100 ml of sterile water per transformation

Prepare electroporation buffer, place on ice, and store at 4°C until ready to use.

• 100 ml of electroporation buffer per transformation.

Electroporation Buffer (500 ml):

• 250 ml 2M Sorbitol
• 5 ml 100 mM CaCl₂
• 150 ml ddH₂O

Prepare conditioning buffer, and store at room temperature until ready to use.

• 20 ml of conditioning buffer per transformation.

Conditioning Buffer (100 ml) :

• 50 ml sterile ddH₂O
• 5 ml 2M LiOAc
• 1 ml 1M DTT
• 44 mL sterile ddH₂O Note: Add in order, as precipitation will occur if undiluted LiOAc and DTT are added together.

Split yeast culture into two 50-ml conical tubes, collect cells by gentle centrifugation at 1500 rcf for 3 minutes, and decant the media.

Resuspend (wash) each cell pellet by gently pipetting with 25-ml ice-cold sterile water , centrifuge at 1500 rcf for 3 minutes, and decant the media.

Wash the cell pellets again with 25-ml ice-cold sterile water , centrifuge at 1500 rcf for 3 minutes, and decant the media.

Wash the cell pellets with 25-ml of ice-cold electroporation buffer , centrifuge at 1500 rcf for 3 minutes, and decant the media.

Resuspend the cell pellets in 10-ml conditioning buffer and pool into a single 125-ml flask. Incubate at 30°C with 270 rpm orbital shaking for 30 minutes.

Pour cells into a 50-ml conical tube, centrifuge at 1500 rcf for 3 minutes, and decant the media.

Note: follow institutional waste disposal procedures for unused or ‘spent’ conditioning buffer.

Wash the cells with 50-ml ice-cold electroporation buffer , centrifuge at 1500 rcf for 3 minutes, and decant the media.

Resuspend the cell pellet to a final volume of 1 ml by adding 100-200 µl electroporation buffer.

• If you need to add more or less electroporation buffer than this, you likely need to optimize your initial inoculation OD₆₀₀.

Keep cells on ice until electroporation.

Pre-chill electroporation cuvette(s) on ice.

• 1 cuvette per transformation.

Prepare Outgrowth Media.

• 8 ml per sample, aliquoted into 50-ml conical tubes.

Outgrowth Media:

• 50 ml 2x YPD
• 50 ml 1M Sorbitol

For each electroporation, transfer 1 µg of DNA and 400 µl of concentrated cells to a microcentrifuge tube and gently mix by pipetting.

Pipet concentrated cells plus DNA into a pre-chilled Gently electroporation cuvette and keep on ice for 5 minutes.

Electroporate the cells with the following instrument settings:

• Voltage: 2.5 kV

• Capacitance: 25 µF

• Resistance: 200 Ohms

• Typical time constant ranges from 3.0 to 4.5 milliseconds.

Using a sterile Pasteur pipette, transfer electroporated cells into the 50-ml conical tube containing 8 ml of outgrowth media.

• To reduce the amount of cells left in cuvette, ~500 µl of the outgrowth media can be pipetted into the cuvette and then transferred back into the 50-ml conical with the rest of the outgrowth media.

Incubate the 50-ml conical tube containing the cells plus 8-ml conditioning media at 30°C without shaking for 1 hour, mixing every 15 minutes by gentle inversion.

• Orbital shaking reduces transformation efficiency, possibly due to increased fragility of electroporated cells.

Following outgrowth, collect cells by gentle centrifugation at 1500 rcf for 3 minutes, and decant the media.

For auxotrophic marker selection, resuspend cells in the appropriate yeast dropout media (e.g., for uracil selection, resuspend cells in SC -Ura). For drug marker selection, resuspend cells in YPD.

For auxotrophic marker selection, plate directly on selective media. For drug marker selection, can either plate directly to selective media, or can plate to YPD and replica print to selective media the next day.

• Replica printing often increases the number of transformants recovered when using drug markers.
• Alternatively, can perform selection in liquid or semi-solid (low-percentage agarose) media and then pellet the transformants.
• To determine the total number of transformants (e.g., if plating to confluency or performing a liquid or semi-solid selection), plate 100 µl of 10-fold serial dilutions down to a 10^-5 dilution.