• Frozen competent yeast cells:
1. Grow cells overnight in 40 ml YPD until an OD660= 0.3-1.2. Harvest cells by by centrifugation in SS34 tubes (2500g x 1 min.) at room temperature.
3. Wash once with 25 ml of sterile water at room temperature.
4. Resuspend pellet with 1 ml of SORB and transfer to 1.5 ml epp. tube. Spin down at 13000 rpm 10 seconds. Remove all supernatant by pipetting out.
5. Resuspend cells in 350 μl of SORB. Add 50 μl of carrier DNA* (0ºC). Mix well.
6. Place the tube at -80ºC.
*Denatured salmon sperm DNA.
• Transformation:
1. Place no more than 10 μl of DNA (5 μg PCR product or 1 μg of plasmid) into one aliquot of competent cellsthawed on ice.
2. Mix well and add a 6-fold volume of PEG solution. Mix throughout andincubate at RT for 30 min.
3. Add 1/9 volumes of DMSO (10% final concentration). Mix well.
4. Heat shock in a water bath at 42ºC for 15 min.
5. Harvest cells by a 2000 rpm x 3 min. centrifugation. Remove allsupernatant.
6. Resuspend in 150 μl of liquid minimal media and plate onto thecorresponding plate. Note: If the selective media is G418, hygromycin or nourseothricin, the pellet must beresuspended in YPD and incubate for at least 3h at 25ºC or more.
• SORB: 100 mM LiAc, 10 mM Tris-HCl pH 8, 1 mM EDTA, 1 mM sorbitol.
• PEG: 100 mM LiAc, 10 mM Tris-HCl pH 8, 40% PEG3350.