Transformation of S cerevisiae by the LiOAc method

Plasmid DNA can be introduced into yeast by several different means. We routinely transform S. cerevisiae using the LiOAc method (74). The transformation efficiency achieved with this method can be influenced by multiple factors. Strain background, cell density at the time of transformation, the quality of the plasmid DNA and carrier DNA used for transformations, and the length of heat-shock can all play some role in influencing the final outcome. When using the protocol below, the transformation efficiency we routinely obtain is 103—104 transformants/ jxg DNA.

Protocol 33.

Method

1. Inoculate a single colony into 50 ml YPD or SD medium in a 250-ml Erlenmeyer flask and grow yeast to a mid-exponential phase (OD600=0.8) at 30°C in an incubator-shaker set at 250 rpm.

2. Harvest cells by centrifugation (5 min at 1000 g) and wash once in 50 ml distilled H20.

3. Collect cells by centrifugation as above. Wash cells in 10 ml LiOAc solution (0.1 M LiOAc, 10 mM Tris-HCI, pH 8.0, 1 mM Na2EDTA) and resuspend the cell pellet in 0.5 ml (1/100 of initial culture volume) LiOAc solution. Cells can be stored in LiOAc at 4°C for up to one week and transformation can be performed at a later time, but with reduced efficiency.

4. Aliquot 100 jxl cell suspension into each microfuge tube and add 5 jjlI 10 mg/ml carrier DNA (sheared single-strand salmon sperm DNA; Sigma) and 1 mg plasmid DNA to each aliquot. Keep the total volume of DNA below 10 ml.

5. Add 0.7 ml PEG solution [40% polyethylene glycol (PEG 3300-4000), 0.1 M LiOAc, 10 mM Tris-HCI, pH 8.0, 1 mM Na2EDTA, pH 8.0] to the cell DNA mixture, vortex vigorously, and incubate at 30°C for 45 min with constant shaking.

6. Heat-shock in a 42°C water bath for 5-15 min. Centrifuge in a microcentrifuge at 1000 g for 5 min and resuspend the cells in 200 |jlI TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM Na2EDTA, pH 8.0).

7. Spread the transformation mixture on SD solid plates lacking amino acids required for selection of the plasmid and place the plates in a 30°C incubator (VWR Scientific Products).

8. Colonies should appear in 2-4 days.

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