ALKALI-CATION
Yeast Transformation Kit
Protocol
Revision No. 2200-999-6D01W

The Alkali-Cation Yeast Transformation Kit provides all of the reagents required to perform an easy and quick non-electrical method for transforming yeast with linear or plasmid DNA. Host cells are made competent by treatment with a lithium/cesium acetate mixture. The entire procedure may be completed in less than 90 minutes and routinely provide a transformation efficiency of greater than 104 transformants per µg of DNA using YEP24 plasmid DNA.

1. Inoculate a single colony into 100 ml YPD (YEPD) Broth (BIO 101 catalog No. 4001-021) and grow with aeration at 30°C to mid log, 2 x 106 to 2 x 107 cells/ml. (Approximately 12-24 hours).



2. Spin to pellet cells at 400 x g for 5 minutes; discard supernatant.



3. Resuspend cells in a total of 10 ml TE, pH 7.5. Spin to pellet cells and discard supernatant.



4. Gently resuspend cells in 5 ml Lithium/Cesium Acetate Solution.



5. Incubate at 30°C for 30 minutes with gentle shaking.



6. Spin at 400 x g for 5 minutes to pellet cells and discard supernatant.



7. Gently resuspend in 1 ml TE, pH 7.5. Cells are now ready for transformation.



8. In a 1.5 ml tube combine:
   
   • 100 µl yeast cells
   • 5 µl Carrier DNA
   • 5 µl Histamine Solution
   • 0.01-1 µg plasmid DNA in a 10 µl volume (max)
   Gently mix and incubate at room temperature for 15 minutes.



9. In a separate tube, combine 0.8 ml PEG and 0.2 ml TE/Cation MIXX for each transformation reaction. Add 1 ml of this PEG/TE/Cation MIXX to each transformation reaction. Mix cells into solution with gentle pipetting.



10. Incubate at 30°C for 10 minutes.



11. Heat shock at 42°C for 10 minutes; cool to 30°C.



12. Pellet cells in a microcentrifuge on high power for 5 seconds and remove supernatant.



13. Resuspend in 200 µl SOSy and incubate at 30°C for 30 minutes before plating on selective media, directly or in top agar (same as plate media but with 1/2 the agar) at <50°C. Incubate at 30°C for 48-72 hours until transformant colonies appear.