Bio-Rad Gene Pulser Xcell™ Electroporation Systems User Manual
Page 72
5.
Add 100 ml of sterile, YPD/HEPES to each of the bottles and vortex to resuspend the cell pellets;
add 2.5 ml of 1M DTT; mix gently. Incubate the cells for 15 min at 30°C without shaking.
6.
Bring the volume to ~500 ml with sterile, ice-cold 1 M sorbitol. Pellet the cells by centrifugation at
3000 x g for 5 min at 4°C; pour off and discard the supernatant.
7.
Add ~50 ml of sterile, ice-cold 1 M sorbitol and vortex to resuspend the cell pellets; bring the
volume to 500 ml with sterile, ice-cold 1 M sorbitol. Pellet the cells by centrifugation at 3000 x g for
5 min at 4°C; pour off and discard the supernatant.
8.
Resuspend the cell pellet in ~25 ml of sterile, ice-cold 1 M sorbitol and transfer to a chilled
30 ml Oakridge tube. Pellet the cells by centrifugation at 3000 x g for 5 min at 4°C; pour off and
discard the supernatant.
9.
Resuspend the cell pellet in 0.5 ml of sterile, ice-cold 1 M sorbitol; the final cell volume should be
~1.3 ml and the cell concentration should be >1 x 10
10
cells/ml. Keep the cells on ice and use as
soon as possible for electroporation.
7.3.2 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 DNA samples (up to 0.1 µg) to be electroporated into sterile 1.5 ml microfuge tubes.
Place tubes on ice.
2.
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 P. pastoris, press 5 or the Down Arrow to highlight P. pastoris, then press Enter to
open the P. pastoris Protocol Detail screen. See Section 3.4 for operating instructions.
4.
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.
5.
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.
6.
Check and record the pulse parameters. The time constant should be close to 5 milliseconds. The
voltage should be approximately 2.0 kV.
7.
When selecting for complementation of an auxotrophic mutant, the cells may be plated
immediately onto minimal agar plates lacking the appropriate nutrient. When selecting for antibiotic
resistance, incubate the cells at 30°C for 1 hr without shaking; add 1 ml of 1M sorbitol and
continue incubating for 1 hr; plate aliquots of the electroporated cells on YPD agar plates with the
appropriate antibiotic. Incubate the plates for 72–96 hrs at 30°C.
7.3.3 Solutions and Reagents
1.
YPD: 10 g yeast extract, 20 g peptone, dissolve in 900 ml water. Autoclave. Add 100 ml sterile
20% glucose.
2.
YPD/HEPES: 100 ml YPD media, 20 ml 1 M HEPES, pH 8.0.
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.
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