TITLE: Electroporation Transformation Protocol in S. Cerevisiae
AUTHORS: Stephanie Hood

[DESCRIPTION]
Protocol adapted by Stephanie Hood, 09/2022, from: 

An improved yeast transformation method for the generation of very large human antibody libraries

Overview:

This protocol calls for ~800-900 ul of cell pellet and is optimized for 1 ug of plasmid DNA in the lab strain (BY4742).


[STEPS]
SECTION: 2-3 Days before starting experiment
1. Streak out yeast strains for electroporation protocol.
Depending on how many cultures you are setting up you may need to streak more than 1 plate to get enough isolated colonies. Grow at 30°C for 2 days.
SECTION: Day before experiment
2. Grow 5 ml culture(s) of S. cerevisiae cells overnight to stationary phase (OD600 to or about 3) in 1XYPD media on a platform shaker at 270 rpm and 30°C.
Need enough cells to set up 100 ml culture(s).
SECTION: Day of experiment: Preparation of electrocompetent cells
3. Turn on spectrophotometer (cuvette) 
SECTION: Day of experiment: Preparation of electrocompetent cells
4. If you have multiple tubes from the same strain, pool the 5 mL O/N cultures into a sterile 50 mL conical tube
Vortex to mix
SECTION: Day of experiment: Preparation of electrocompetent cells
5. Spec the O/N cells 
1:50 dilution (20 ul cells: 980 ul 1X YPD) 
SECTION: Day of experiment: Preparation of electrocompetent cells
6. Set up the culture(s) in 500 mL flasks.

Example: 
100 ml 2X YPD culture — starting OD600 of 2.0
SECTION: Day of experiment: Preparation of electrocompetent cells
7. Use C1V1=C2V2 to calculate the amount of O/N cells you need to add to 100 ml for a starting OD600 of ______. 
Grow the inoculated cultures on a platform shaker at 30°C and 270 rpm for 4 hours.
SECTION: While cultures are incubating: prepare media
8. Put sterile water on ice and place at 4°C.
~100 mL of sterile water needed per reaction.  
SECTION: While cultures are incubating: prepare media
9. Prepare electroporation buffer and put in ice at 4°C.


500 mL Electroporation buffer: ~100 mL needed per reaction
Add 250 mL 2M Sorbitol to a sterile 500 mL bottle
Add 5 mL 100 mM CaCl2
Bring volume to 500 mL with sterile water.


SECTION: After 4 hour incubation
10. Collect yeast cells by centrifugation at 1500 g for 3 minutes and remove the media.
2-50 ml conical tubes - 50 ml each 
SECTION: After 4 hour incubation
11. Prepare conditioning buffer during spins- 0.1 M LiAc/10 mM DTT.

Conditioning Buffer: 20 mL needed per reaction
Add 50 mL sterile water to a sterile 250 mL sterile bottle
Add 5 mL 2M Lithium Acetate
Add 1000 ul 1M DTT
Bring volume to 100 mL (44 mL sterile water added with a serological pipette)

Put any remaining conditioning buffer in a well labeled 50 mL conical tube for disposal with EHS. 
SECTION: After 4 hour incubation
12. Wash the cell pellets with 25 mL ice-cold water.
50 ml conical tubes - 25 ml each
Spin at 1500 g for 3 minutes and remove the media.
SECTION: After 4 hour incubation
13. Wash the cell pellets again with 25 mL ice-cold water.
50 ml conical tubes - 25 ml each
Spin at 1500 g for 3 minutes and remove the media.
SECTION: After 4 hour incubation
14. Wash the cell pellet with 25 mL of ice-cold electroporation buffer (1M Sorbitol / 1 mM CaCl2)
50 ml conical tubes - 25 ml each
Spin at 1500 g for 3 minutes and remove the media.
SECTION: After 4 hour incubation
15. Condition the yeast cells by re-suspending the cell pellet in 10 mL 0.1 M LiAc/10 mM DTT.
50 ml conical tubes - 10 ml each
Pool the tubes culture into one flask for the outgrowth.
10 ml of conditioning buffer for each tube, then pool in a 125 mL flask.
Shake at 270 rpm in a culture flask for 30 minutes at 30 °C.
SECTION: After 4 hour incubation
16. Pour cells into a 50 mL conical tube.
Collect the conditioned cells by centrifugation (1500g for 3 minutes).
SECTION: After 4 hour incubation
17. Wash the cells once with 50 mL ice-cold electroporation buffer.
Spin at 1500 g for 3 minutes and remove the media.
SECTION: After 4 hour incubation
18. Re-suspended the cell pellet in 100 to 200 μL electroporation buffer to reach a final volume of 1 mL.
Record the volume needed to get to 1 mL


Example: 700 ul cell pellet + 300 ul electroporation buffer = 1 mL
2 electroporation reactions of 400 μl each.

You may need to add more electroporation buffer to reach 1 mL cell mixture. 
If only doing 1 transformation, less volume can be added as only 400 ul is needed per transformation. 
SECTION: After 4 hour incubation
19. The cells are kept on ice until electroporation.
SECTION: DNA preparation and electroporation and plating
20. Pre-chill BioRad GenePulser cuvette(s) (0.2 cm electrode gap).
1 cuvette per transformation 
SECTION: DNA preparation and electroporation and plating
21. Prepare enough outgrowth media for all your samples:  1:1 1M sorbitol: 2X YPD

8 mL per sample aliquoted into 50 ml conical tubes.
SECTION: DNA preparation and electroporation and plating
22. Use 1 ug of plasmid DNA per 400 ul of cells.
Add ____ uL of plasmid DNA to the 1 mL of cells = 1 ug per 400 uL.  
2.5 ug of plasmid DNA in 1 mL of cells.
SECTION: DNA preparation and electroporation and plating
23. Gently mix 400 μl electrocompetent cells and DNA and transfer to a pre-chilled BioRad GenePulser cuvette (0.2 cm electrode gap). 
Kept on ice for 5 minutes until electroporation.
SECTION: DNA preparation and electroporation and plating
24. Electroporate the cells at 2.5 kV and 25 μF. 
Typical time constant ranges from 3.0 to 4.5 milliseconds.
200 Ohms
SECTION: DNA preparation and electroporation and plating
25. Using a sterile pasteur pipette, transfer electroporated cells from each cuvette into 8 mL of 1:1 mix of 1 M sorbitol : 2X YPD media. 
SECTION: DNA preparation and electroporation and plating
26. Incubate the 50 mL conical tube containing the 8 mL of recovery media in the 30°C standing incubator for 1 hour.
Mix by gentle inversion (~4x) every 15 mins.
Use a rack to store 50 mL conical tubes in the incubator. Do not store conical tubes upside down as they tend to leak.
SECTION: DNA preparation and electroporation and plating
27. Collect cells by centrifugation (1500g for 3 minutes) and resuspend in SD URA- + 2% glucose. 
Use 1 mL SD URA- + 2% glucose media for every 400 μl electroporated cells.
SECTION: DNA preparation and electroporation and plating
28. Prepare 10-fold serial dilutions from the cell suspension and plate 100 μl of 10-2, 10-3, and 10-4 dilutions onto selection plates and incubate at 30 °C incubator for 2 days.
If using an antibiotic marker for selection, let the cells grow on just YPD for 1 days and then replica print onto YPD + antibiotic the following day. 
SECTION: 2-3 days after experiment
29. Count the number of colonies on each plate to determine the CFUs.