Yeast Transformation Using Frozen Competent Cells and Single-stranded DNA as a Carrier
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Yeast Transformation Using Frozen Competent Cells and Single-stranded DNA as a Carrier
Katherine Kolor
References:
- Schiestl and Gietz (1989) Curr. Genetics 16:339-346
- Gietz, et al (1995) Yeast 11:355-360
Preparing 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 23o C for 1.0 - 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 -70o C 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 microliters of herring sperm DNA (8 mg/ml) in an eppendorf tube for 15 min. Cool on ice 5 min.
2. Add transforming DNA (up to 5 micrograms) to the carrier.
3. Add 100 microliters 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 30o C for 30 min.
6. Transfer tube to 42o C water bath, incubate for 15 min.
7. Plate an aliquot of transformation mix directly onto selective medium.
Stock solutions
1X TE-LiAc
PEG-TE-LiAc
