Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 71)

7.2.2 Electroporation
1. For each sample to be electroporated, prepare a 17 x 100 mm sterile tube with 1 ml of 1.2 M
Sorbitol and place on ice; also place a 0.2 cm cuvette on ice.
2. Pipette the DNA samples (up to 1 µg) to be electroporated into sterile 1.5 ml microfuge tubes.
Place tubes on ice. Add 40 µl of the competent cells to each DNA sample and mix gently.
3. From the Home Screen on Gene Pulser Xcell open the Fungal Protocol Screen (press 4, Enter, 2,
Enter). To select S. pombe, press 3 or the Down Arrow to highlight S. pombe, then press Enter to
open the S. pombe Protocol Detail screen. See Section 3.4 for operating instructions.
4. Transfer the DNA - cell samples to the chilled 0.2 cm electroporation cuvettes and tap the suspension
to the bottom. Place the cuvette in the ShockPod. Push the chamber lid down to close. Pulse
once.
5. Remove the cuvette from the chamber and immediately add 1 ml of ice cold 1.2 M sorbitol to the
cuvette; gently transfer the diluted cells to a sterile 17 x 100 mm tube.
6. Check and record the pulse parameters. The time constant should be close to 5 milliseconds. The
voltage should be approximately 2.3 kV.
7. Incubate the tubes at room temperature for 40–60 min. Plate aliquots of the electroporated cells on
selective agar plates without sorbitol (e.g., EMM). Incubate plates for 6–8 days at 30°C.
7.2.3 Solutions and Reagents
1. YCD media: 10 g yeast extract, 2 g casamino acids, dissolve in 900 ml water. Autoclave. Add
100 ml 20% glucose.
20% Glucose: 100 g glucose, dissolve in 350 ml water; bring volume to 500 ml with water. Filter
sterilize.
2. 1.2 M sorbitol: 218.6 g sorbitol, dissolve in ~700 ml water. Add water to 1.0 L. Autoclave.
3. EMM: 12.35 g EMM – Dextrose (Q-Biogene, Catalog # 4110-122), dissolve in 900 ml water. Pour
450 ml into each of two 1 L bottles and add 10.0 g agar to each bottle. Autoclave 30 min. Cool to
55–60°C and add 50 ml 20% Glucose to each bottle. Pour plates (sufficient for ~60 plates).
7.3 Pichia pastoris
7.3.1 Preparation of Electrocompetent Cells
See Cregg & Russell (1998) for additional information.
Using this method we have obtained transformation efficiencies of ~6 x 10 4 transformants / µg
electroporating P. pastoris X33 with SacI-pPICZA (Invitrogen).
Gene Pulser Xcell conditions: C = 25 uF; PC = 200 ohm; V = 2.0 kV.
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 a fresh overnight culture of
P. pastoris. The doubling time of P. pastoris is approximately 2 hrs at 30°C.
2. Incubate at 30°C overnight, shaking at 300 rpm, to a density of ~7 x 10 7 cells/ml (OD
600
(1:10 dil)
= 0.24–0.30).
3. Decant the cells into a sterile 500 ml centrifuge bottle and pellet the cells by centrifugation at
3000 x g for 5 min at 4°C.
4. Carefully pour off and discard the supernatant.
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