Transformation Protocol

1. Revival:  Take competent cells from -80°C, and immediately put them in a 30 °C water bath for 90 seconds with gentle agitation. Keep the vial on wet ice for 3min.
2. Wash: Add 1.6ml of ice-cold electroporation buffer (1M sorbitol/1mM CaCl2) to the cells, and spin down at 2000xg for 2min at 4°C. Use a pipette to remove all the supernatant and don’t touch the pellet. 
3. DNA Preparation: (i) DNA must be purified, concentrated with ethanol precipitation, and dissolved in water. The presence of salt can lead to arcing at high voltage. (ii) Total DNA volume is less than 30ul. The smaller the volume, the better. (iii) add 5ug of vector and 10ug of inserts (overhangs 50bp) into ~250 ul of electroporation buffer, mix well, and sit on wet ice.
4. Electroporation: Resuspend the cell pellet with ~280ul of DNA/electroporation buffer, and the final volume will be 350-400ul (It is easy to cause arcing if the total volume exceeds 400ul). Transfer the cell/DNA mixture into an ice-cold 2mm electroporation cuvette without introducing bubbles. Electroporation @ 2.5 kV, 25 uF, and 200Ω, and the typical time constant should be > 4.0ms. Immediately add 10 ml of YPD containing 1M sorbitol (Teknova #Y5300). Shake at 225 rpm and 30°C for 2 hours.
5. Calculation of transformants: (i) Serial 10x dilution: add 50ul of cell culture into 450ul of dilution buffer (1X PBS containing 0.05% Tween20). (ii) spread 100ul of diluted cells onto selective agar plates (Teknova # C3060). Incubate at 30°C for 3 days. (iii) calculate transformants: if 100ul of 100000x diluted culture produces 20 colonies, the total transformants are 2.0e+8. 
6. Library growth: Spin down 10ml of cell culture at 2500xg for 3min. Remove the supernatant. Add 200ml of selective growth medium (Teknova # C8130), and 2ml of Penicillin-Streptomycin (Gibco #15140122). Shake at 30°C and 225rpm for 48hours.
7. Library Storage: Spin down 200ml of cell culture and Resuspend the cells in freezing solution (90% YPD medium, 10% glycerol) to give a final cell concentration of ~1.0e+9 cells /ml. Aliquot at 1ml/vial. Place the vials in a bath of isopropanol and slow-freeze the cells at -80°C.
8. Library display: Culture the library in the induction medium (Teknova # C9115) containing 0.2% glucose and Penicillin-Streptomycin. The initial OD600 is 0.5~1.0 and shakes at 225rpm and 20~25 °C for ~24h. The final OD600 should be at least ~4 times higher than the initial OD600. 

DNA ethanol precipitation

1. Add 1/10 volume of 3 M sodium acetate, 1/50 volume of glycogen (Thermo Scientific #R0561), 1 volume of isopropanol (or 2.5 volume of ethanol)to the DNA solution, mix well, and incubate the mixture at -20 °C for > 60 min.
2. Spin at full speed at 4 degrees for 20 min. Discard the supernatant. 
3. Rinse the pellet with 1 ml of 70% ethanol. Spin at full speed at 4 degrees for 5 min and discard the supernatant.
4. Air-dry the pellet. Dissolve DNA in nuclease-free Water.

References

1. Benatuil L, Perez JM, Belk J, Hsieh CM. An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel. 2010; 23(4): 155-9.
2. Suga M, Hatakeyama T. High-efficiency electroporation by freezing intact yeast cells with addition of calcium. Curr Genet. 2003; 43(3): 206-11.

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