Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 74)

4. From the Home Screen on Gene Pulser Xcell open the Fungal Protocol Screen (press 4, Enter, 2,
Enter). To select C. albicans, press 4 or the Down Arrow to highlight C. albicans, then press Enter
to open the C. albicans Protocol Detail screen. See Section 3.4 for operating instructions.
5. Transfer the DNA - cell samples to the 0.2 cm electroporation cuvettes that have been chilled in ice
and tap the suspension to the bottom. Place the cuvette in the ShockPod. Push the chamber lid
down to close. Pulse once.
6. Remove the cuvette from the chamber and immediately add 1.0 ml of ice cold 1.0 M sorbitol to the
cuvette; gently transfer the diluted cells to a sterile 17 x 100 mm tube.
7. Check and record the pulse parameters. The time constant should be close to 5 milliseconds. The
voltage should be approximately 1.5 kV.
8. Immediately plate an aliquot of the cells onto minimal agar plates containing 1 M sorbitol but lack-
ing the appropriate nutrient. Incubate the plates for 4–6 days at 30°C.
7.4.3 Solutions and Reagents
1. YPD / Uridine: 10 g yeast extract, 20 g peptone, dissolve in 900 ml water; add 15 g agar.
Autoclave. Add 100 ml sterile 20% glucose, 1 ml 25 mg/ml uridine.
2. YPD / Uridine agar: 10 g yeast extract, 20 g peptone, dissolve in 900 ml water. Autoclave. Add
100 ml sterile 20% glucose, 1 ml 25 mg/ml uridine.
3. 1M sorbitol: 182.2 g sorbitol, dissolve in 800 ml water. Bring volume to 1.0 L with water.
Autoclave.
4. 20% glucose: 20 g glucose, dissolve in 60 ml water. Adjust volume to 100 ml with water. Sterilize
through a 0.22 µ filter.
5. 5 mM LiAc/10 mM DTT in TE: 1 ml 1 M Lithium Acetate (10.2 g / 100 ml), 20 ml 0.1 M DTT (1.54
g / 100 ml), 2 ml 1 M Tris, pH 7.5, 0.4 ml 0.5 M EDTA, 176.6 ml water. Filter sterilize. Store at
–20°C.
6. 1M sorbitol: 182.2 g sorbitol, dissolve in 800 ml water. Bring volume to 1.0 L with water.
Autoclave.
7.5 Dictyostelium discoideum
7.5.1 Preparation of Electrocompetent Cells
See Howard et al. (1988) and Knecht & Pang (1995) for additional information.
Gene Pulser Xcell conditions: square wave, V = 1.0 kV, 1.0 ms Pulse length, 2 pulses, 5 sec pulse interval;
V = 1.0 kV.
1. Inoculate Dictyostelium cells at a concentration of 5–7 x 10 5 cells/ml into 40 ml of HL5 media in a
500 ml flask. The cells may either be scraped from a plate or transferred from liquid media. The
doubling time of Dictyostelium is approximately 12 hrs at 21°C.
2. Incubate the culture at 21°C for about 24 hrs, shaking at 125 rpm. About 16–20 hrs prior to
preparing the competent cells, dilute the cells to 7 x 10
5 cells/ml with HL5 media. Incubate at 21°C
overnight, shaking at 125 rpm.
3. Transfer 100 ml of the cells into two sterile, disposable, 50 ml centrifuge tubes and incubate on ice
for 15 min to stop growth.
4. Pellet the cells by centrifugation at 400 x g for 5–7 min at room temperature.
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