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Yeast transformation

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 23oC 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 -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 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 30oC for 30 min.

6. Transfer tube to 42oC water bath, incubate for 15 min.

7. Plate an aliquot of transformation mix directly onto selective medium.


Stock solutions

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


Questions? Contact Katie Kolor

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