Yeast Transformation Using Frozen Competent Cells and Single-stranded DNA as a Carrier
Protocol by Katie Kolor based on Schiestl and Gietz (1989) Curr. Genetics 16:339-346; Gietz, et al (1995) Yeast 11:355-360Preparing competent cells:
| 1. | Inoculate 100 ml yeast synthetic complete medium + 3% glycerol medium from overnight culture. Grow culture to 0.5-1.0 x 107 cells/ml. (2-3x higher efficiency if diluted back to 2 x 106 cells/ml, then grown 2-3 more doublings.) Note: The cells must be grown in glycerol if you are going to freeze them. Glucose-grown cells lose competence upon freezing. |
| 2. | Pellet cells gently (~800 x g). Resuspend in 7-8 ml of 1x TE-LiAc. |
| 3. | Pellet cells gently. Resuspend in 1 ml of 1x TE-LiAc. |
| 4. | Rotate at 23°C for 1 - 1.5 hr. |
Cells can be frozen at this point for future use by wrapping the tube in several layers of Kimwipes and/or paper towels and placing them in an ice cream container in a -70oC freezer. It is important to wrap up the tube so that the cells freeze slowly. Thaw cells at room temperature for transformation.
Transformation:
| 1. | Boil 20 µl of herring sperm DNA (8 mg/ml) in an microfuge tube for 15 min. Cool on ice 5 min. |
| 2. | Add transforming DNA (up to 5 µg) to the carrier. |
| 3. | Add 100 µl of competent cells to the DNA. |
| 4. | Add 700 microliters of PEG-TE-LiAc solution (made fresh from the stock solutions). Vortex briefly and gently to mix. |
| 5. | Rotate tube at 30°C for 30 min (23°C for ts strains). |
| 6. | Transfer tube to 42°C water bath, incubate for 15 min. (Can be reduced to 37°C for ts strains.) |
| 7. | Plate an aliquot of transformation mix directly onto selective medium. |
- 50% Polyethylene glycol 4000
- 1 M Tris-HCl pH 8.0
- 1 M Lithium acetate pH 7.5
- 0.2 M EDTA pH 8.0
1x TE-LiAc
- 10 mM Tris-HCl pH 8.0
- 1 mM EDTA
- 0.1 M Lithium acetate
PEG-TE-LiAc
- 40% PEG-4000
- 10 mM Tris-HCl pH 8.0
- 1 mM EDTA
- 0.1 M Lithium actetate
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