Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 73)

5. 1 M HEPES, pH 8.0: 23.8 g HEPES (MW = 238.3), dissolve in ~65 ml water. Adjust pH to 8.0 with
5 N NaOH. Bring volume to 100 ml with water. Sterilize through a 0.22 µ filter.
6. 1M DTT: 3.85 g DTT, dissolve in ~22 ml water. Bring volume to 25 ml with water. Sterilize through
a 0.22 µ filter.
7.4 Candida albicans
7.4.1 Preparation of Electrocompetent Cells
See deBacker, et al. (1999) for additional information.
Using this method we have obtained transformation efficiencies of >400 transformants / µg
electroporating C. albicans CAI4 with NheI-pUX (Thompson, et al., 1998).
Gene Pulser Xcell conditions: C = 25 µF; PC = 200 ohm; V = 1.5 kV.
This procedure requires a Gene Pulser Xcell main unit and PC Module.
1. Inoculate a colony of C. albicans into 5–6 ml of YPD/uridine media in a 17 x 100 mm tube; incubate
overnight at 30°C shaking at 250–300 rpm.
2. Inoculate an aliquot of the overnight culture into 500 ml of YPD/uridine media in a 2.8 L Fernbach
flask; incubate overnight at 30°C shaking at 250–300 rpm to a density of 5–10 x 10
7 cells/ml
(OD
600
of a 1:10 dilution = 0.3–0.45).
3. Chill the culture on ice for 10 min.
4. Transfer the cells into a sterile 500 ml centrifuge bottle.
5. Pellet the cells by centrifugation at 15–20°C for 5 min at 3000 x g.
6. Discard the supernatant. Resuspend the cell pellet in 125 ml of sterile 5 mM LiAc/10 mM DTT;
incubate at 20°C for 1 hr.
7. Pellet the cells at 4°C for 5 min at 3000 x g.
8. Discard the supernatant. Resuspend the pellet in 125 ml of sterile, ice cold water. Pellet the cells
as above.
9. Discard the supernatant. Resuspend the cell pellet in 125 ml of ice cold water. Pellet the cells as
above.
10. Discard the supernatant. Resuspend the cell pellet in 35 ml of ice cold 1 M sorbitol. Transfer the
cells to a chilled Oakridge tube. Pellet the cells at 4°C for 5 min at 3000 x g.
11. Carefully remove the supernatant. Resuspend the pellet in 0.5 ml of ice cold 1 M sorbitol.
12. Keep the cells on ice and use as soon as possible for electroporation.
7.4.2 Electroporation
1. For each sample to be electroporated, prepare a 17 x 100 mm sterile tube with 1.0 ml of 1 M
sorbitol and place on ice; also place a 0.2 cm cuvette on ice.
2. Pipette the linearized DNA samples (0.1–1 µg) to be electroporated into sterile 1.5 ml microfuge
tubes. Place tubes on ice.
3. Add 40 µl of the electrocompetent cells to each DNA sample and mix gently. Incubate the cells on
ice for 5 min.
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