Yeast Transformation II
Low Efficiency Transformation (for Plasmids)
Inoculate 5 mls per transformation. Grow yeast overnight (use YPD if possible, selective media if necessary).
High Efficiency (for Linear DNA or Libraries)
Inoculate 5 mls. Grow yeast overnight. Inoculate 50 mls YPD to OD600 0.07. Grow for 4-5 doubles (OD600 0.6-1.0).
For Both
- Harvest cells. Spin at 2500 rpm for 5 minutes, and discard media.
- Wash once with sterile H2O.
- Resuspend in 1-3 mls sterile H2O, transfer to Eppendorf tube, spin down, discard supernatant.
- Resuspend in 1X lithium acetate/TE, spin down, discard supernatant.
- Add ~1 µg (in 1-8 µl) transforming DNA and 10 µl of single stranded carrier DNA. Mix gently.
- Add 600 µl PEG/lithium acetate/TE solution, and mix gently.
- Incubate with gentle rotation at 30°C for 30 min.
- Heat shock at 42°C for 15 min.
- Add sterile H2O, and wash yeast 2-3 times.
- Resuspend in 500 µl of H2O.
- Plate on appropriate selective media.
Solutions
Carrier DNA
- 10 mg/ml
- Boil 5 min
- Place on ice for 5 min before use
10X Lithium Acetate
- 1M, pH 7.5
10X TE
- 100mM Tris, pH 7.5
- 10mM EDTA
- Filter sterilize
50% PEG (3500-4000)
- Dissolve in water
- May have to heat to dissolve
1X Lithium Acetate/TE
- 100 µl 10X TE
- 100 µl 10X LiAc
- 800 µl H2O
PEG/Lithium Acetate/TE
- 960 µl 50% PEG
- 120 µl 10X LiAc
- 120 µl 10X TE