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The Laboratory of Jasper Rine at the University of California, Berkeley
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1. Inoculate a single colony of the desired yeast strain to be transformed into 5 ml YPD and grow overnight at the appropriate temperature.
2. From the above overnight culture, inoculate into 200 ml YPD the appropriate amount to make the culture's optical density at 600 nm (OD600) less than or equal to 0.1.
3. Place 100 ml of inoculated culture into each of two 500 ml flasks.
4. Shake at the appropriate temperature until OD600 is between 0.6 and 1.8.
5. Pellet cells 5 min at 3,000 rpm in a table top centrifuge at room temperature.
6. Resuspend the pellet in 20 ml TEL Buffer.
7. Shake the cells at room temperature for 20 to 30 min.
8. While cells are shaking, aliquot 2 μl of 10 mg/ml salmon sperm DNA to each of 96 wells in a microtiter plate.
9. Add 1 to 10 μl of desired plasmid DNA to be transformed to each appropriate well.
10. Pellet cells and resuspend into a total volume of 3 ml TEL.
11. Aliquot 28 μl of cell suspension into each of the 96 wells in the microtiter plate. Mix by pipetting up and down.
12. Incubate at room temperature for 30 min.
13. Add 196 μl of 40% PEG/TEL Buffer to each well.
14. Incubate at room temperature for 30 min.
15. Heat shock by placing in a 42°C heating block for 5 to 10 min.
16. Pellet the cells by centrifuging at 2500 rpm for 5 min in a swinging bucket rotor centrifuge with adapters for 96-well plates.
17. Remove 40% PEG/TEL Buffer and resuspend cells in 28 μl of TE (see Hint #1).
18. Use either a 48 or 96 pin "frogger" to imprint a grid on the appropriate selective plates.
19. Plate 5 μl of each cell transformation mix onto the corresponding spot on the grid. Allow the liquid to absorb into the plate before moving.
20. Incubate at the appropriate temperature for 2 to 3 days.
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| 40% PEG/TEL Buffer |
| Make fresh for each use
4 parts 50% (w/v) PEG 4000 plus 1 part 5X TEL
Mix by pipetting up and down
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| TEL Buffer |
| 1 mM EDTA
10 mM Tris, pH 7.5
0.1 M Lithium Acetate
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| YPD |
| Autoclave 20 min, cool to room temperature
10 g/liter Yeast Extract
20 g/liter Glucose (Dextrose)
20 g/liter Peptone
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| TE Buffer |
| 1 mM EDTA
10 mM Tris, pH 7.5
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Tris
Polyethylene Glycol (PEG) 4,000
EDTA
Peptone
Lithium Acetate
Glucose
Yeast Extract
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1. Instead of resuspending the cells in 28 μl of TE, you can transfer them directly to selective plates using either the 48 or 96 pin "frogger". This will result in a reduced yield of transformants but will be adequate for most applications.
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