How to Make and Transform Competent Yeast Cells
Competence is a special physiological state of a cell in which a cell can take up exogenous DNA from the surrounding environment. Using lithium acetate and polyethylene glycol (PEG), yeast cells can be made competent for transformation and subsequent downstream applications.
In this method, lithium acetate changes cell permeability to allow uptake of foreign DNA. To protect yeast cell membrane from the high concentration of lithium acetate and allow the plasmid to contact the cell membrane more tightly for an efficient transformation, PEG is added to the transformation solution. The squid sperm monomer DNA (ssDNA), a short linear single-stranded DNA, can also be added to the transformation cocktail to prevent DNA degradation by yeast DNase and also to promote the absorption of foreign DNA.
Protocol for Making Yeast Competent Cells
Reagents
- Yeast cells
- YPD medium (1% yeast extract,2% bactopeptone, 2% dextrose)
- YPD agar plates
- Sterile ddH2O
- Sterile glycerol (10%)
- Lithium acetate (LiAc, 0.1 M)
Equipment
- Shaker
- Stationary and shaking incubator
- Refrigerated centrifuge
- Ice bucket with ice and liquid nitrogen
- Tubes (centrifuge, test, Eppendorf)
