Transformation Protocol

1. Revival: Take competent cells from -80°C, and immediately put them in a 30 °C water bath for 15 seconds with gentle agitation. Keep the vial on wet ice for 3min.
2. Wash: Add 1.4ml of ice-cold electroporation buffer (1M sorbitol/1mM CaCl2) to the cells, mix well, 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 300ng of linearized vector and 600ng of inserts (overhangs 50bp) into ~20 ul of electroporation buffer, mix well, and sit on wet ice.
4. Electroporation: Resuspend the cell pellet with ~20ul of DNA/electroporation buffer, and the final volume will be 30~40ul. 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 ~5.0ms. Immediately add 1 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 10000x diluted culture produces 20 colonies, the total transformants are 2.0e+6. 

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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