TITLE: High-Efficiency Yeast Electroporation
AUTHORS: Stephanie Hood, Jeffrey A Lewis

[DESCRIPTION]
For when extremely high transformation efficiencies are needed (e.g., for transforming plasmid DNA libraries). Adapted from An improved yeast transformation method for the generation of very large human antibody libraries.

Overview:
Using strain BY4742, transformation efficiencies were optimal using 400 µl of concentrated cells and 1 µg of plasmid DNA, yielding ~3.5 million transformants per electroporation. It is possible that other strains may have different optima. The protocol is designed to allow for 2 electroporations per culture (yields ~1ml of concentrated cells) and can be scaled up for producing libraries. 


[GUIDELINES]
The OD600 values are based on those obtained from a Unico 1100RS Spectrophotometer.

[STEPS]
SECTION: 2 days before starting experiment
1. Streak out yeast strains to be transformed onto YPD agar and grow at 30°C for 2 days.
SECTION: Day before experiment
2. 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.

SECTION: Day of experiment: preparation of electrocompetent cells
3. Pool the individual 5-ml overnight cultures into a sterile 50-ml conical tube and vortex to mix.
SECTION: Day of experiment: preparation of electrocompetent cells
4. Measure the OD600 of the saturated cells.
Generally need to make a 1:20 or 1:50 dilution to be within the linear range of the spectrophotometer.
SECTION: Day of experiment: preparation of electrocompetent cells
5. In a 500-ml flask, dilute cultures to be transformed to a starting OD600 of 2.0 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.
SECTION: Day of experiment: preparation of electrocompetent cells
6. Grow the 100-ml inoculated cultures at 30°C with 270 rpm orbital shaking for 4 hours.
SECTION: While cultures are incubating: prepare media
7. Place sterile water on ice and store at 4°C until ready to use.
100 ml of sterile water per transformation
SECTION: While cultures are incubating: prepare media
8. 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
245 ml ddH2O
SECTION: After 4 hour incubation
10. Split yeast culture into two 50-ml conical tubes, collect cells by gentle centrifugation at 1500 rcf for 3 minutes, and decant the media. 

SECTION: After 4 hour incubation
11. 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.

SECTION: After 4 hour incubation
12. Wash the cell pellets again with 25-ml ice-cold sterile water, centrifuge at 1500 rcf for 3 minutes, and decant the media.
SECTION: After 4 hour incubation
13. Wash the cell pellets with 25-ml of ice-cold electroporation buffer, centrifuge at 1500 rcf for 3 minutes, and decant the media.
SECTION: After 4 hour incubation
14. 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.
SECTION: After 4 hour incubation
15. 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.
SECTION: After 4 hour incubation
16. Wash the cells with 50-ml ice-cold electroporation buffer, centrifuge at 1500 rcf for 3 minutes, and decant the media.
SECTION: After 4 hour incubation
17. 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. 
SECTION: After 4 hour incubation
18. Keep cells on ice until electroporation.
SECTION: Electroporation, outgrowth, and plating
19. Pre-chill electroporation cuvette(s) on ice.
1 cuvette per transformation.
SECTION: Electroporation, outgrowth, and plating
20. Prepare Outgrowth Media.
8 ml per sample, aliquoted into 50-ml conical tubes.

Outgrowth Media:
50 ml 2x YPD
50 ml 1M Sorbitol
SECTION: Electroporation, outgrowth, and plating
21. For each electroporation, transfer 1 µg of DNA and 400 µl of concentrated cells to a microcentrifuge tube and gently mix by pipetting. 
SECTION: Electroporation, outgrowth, and plating
22. Pipet concentrated cells plus DNA into a pre-chilled Gently electroporation cuvette and keep on ice for 5 minutes. 
SECTION: Electroporation, outgrowth, and plating
23. 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.
SECTION: Electroporation, outgrowth, and plating
24. 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.
SECTION: Electroporation, outgrowth, and plating
25. 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. 
SECTION: Electroporation, outgrowth, and plating
26. Following outgrowth, collect cells by gentle centrifugation at 1500 rcf for 3 minutes, and decant the media. 
SECTION: Electroporation, outgrowth, and plating
28. 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.
SECTION: While cultures are incubating: prepare media
9. 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.
SECTION: Electroporation, outgrowth, and plating
27. 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.