Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 68)

5. Immediately after the pulse place the cuvette on ice for 30 min. Add 1 ml MRS/SG to the cuvette
and transfer the plasmid/cell suspension to a sterile, 17 x 100 mm tube. Incubate the cells for
3–4 hrs at 30°C without shaking.
6. Plate onto MRS agar plates containing antibiotic.
7. Incubate 2–3 D at 30°C.
6.7.3 Solutions and Reagents
1. 2X MRS (w/o Glucose): 10 g Proteose Peptone No 3 (Difco #211693), 10 g Beef Extract (Difco
#211520), 5 g Yeast Extract (Difco #212750), 1 ml Tween 80, 11.5 ml 1 M K
2
HPO
4
, 12.3 ml 3 M
Na Acetate, 1.9 g citric acid, 0.8 ml 1 M MgSO
4
, 225 µl 1 M MnSO4. Dissolve in water and bring
the volume to 500 ml. Autoclave
2. 1 X MRS: 500 ml 2 X MRS, 200 ml 10% Glucose (filter sterilized), 300 ml sterile water
3. 1 X MRS Agar with antibiotic: To a 1 L bottle, add 7.5 g Bacto agar and 150 ml water;
autoclave. Cool to 55°C. Warm 250 ml 2 X MRS and 100 ml 10 % Glucose to 55°C; add to the
agar. Add antibiotic. Pour plates.
4. MRS/SG (MRS with 0.75 M Sorbitol + 1% Glycine): 250 ml 2 X MRS, 50 ml 20 % Glucose (sterilized
by filtration), 150 ml sterile 2.5 M Sorbitol, 33 ml sterile 2 M Glycine, 17 ml sterile water.
5. 2 M Glycine: 15.0 g in 100 ml water; filter sterilize.
6. 2.5 M Sorbitol: 227.8 g Sorbitol; dissolve in ~250 ml water then bring volume to 500 ml. Filter
sterilize.
7. 30% PEG 1000: 150 g PEG 1000 (warm to ~50°C to melt; pour into beaker with ~200 ml water).
Bring volume to 500 ml with water. Filter sterilize.
Section 7
Electroporation of Fungal Cells
7.1 Saccharomyces cerevisiae
7.1.1 Preparation of electrocompetent cells
See Becker & Guarante (1991) and Ausubel et al. (1987) for additional information. Using this procedure
we have obtained >5 x 10
5 transformants/µg electroporating S. cerevisiae Sc948 with Yep352.
Gene Pulser Xcell conditions: C = 25 µF; PC = 200 ohm; V = 1.5 kV (in 0.2 cm cuvettes), or
C = 25 µF; PC = 200 ohm; V = 2.5 kV (in 0.4 cm cuvettes).
This procedure requires a Gene Pulser Xcell main unit and PC Module.
1. Inoculate 500 ml of YPD in a 2.8 L Fernbach flask with an aliquot from an overnight culture of
S. cerevisiae. The doubling time of S. cerevisiae is approximately 2 hrs at 30°C.
2. Incubate at 30°C overnight, shaking at 250 rpm, to a density of ~ 1 x 10 8 cells/ml (OD
600
(1:10
dilution) = 0.30–0.35).
3. Chill the cells in an ice water bath for 15 min to stop growth.
4. Decant the cells into a sterile 500 ml centrifuge bottle and pellet the cells by centrifugation at
4000 x g for 5 min at 4°C.
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