Yeast Transformation Using Frozen Competent Cells and Single-stranded DNA as a Carrier

Yeast Transformation Using Frozen Competent Cells and Single-stranded DNA as a Carrier

Katherine Kolor

 

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References:

 

Schiestl and Gietz (1989) Curr. Genetics 16:339-346

Gietz, et al (1995) Yeast 11:355-360

 

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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 23^o 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 -70^o 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 30^o C for 30 min.

6. Transfer tube to 42^o C water bath, incubate for 15 min.

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

 

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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