Bio-Rad Gene Pulser Xcell™ Electroporation Systems User Manual
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5.
Carefully pour off and discard the supernatant; place the centrifuge bottles with the cell pellets on
ice.
6.
Add 80 ml of sterile water to the bottle and vortex to resuspend the cell pellet.
7.
Add 10 ml of 10 X TE buffer; swirl to mix. Add 10 ml of Lithium Acetate stock solution; swirl to mix.
Incubate 45 min at 30°C shaking at ~85 RPM.
8.
Add 2.5 ml of 1 M DTT; swirl to mix. Incubate 15 min at 30°C shaking at ~85 RPM.
9.
Bring the volume to 500 ml with sterile water. Pellet the cells by centrifugation at 4000 x g for 5 min
at 4°C; pour off and discard the supernatant.
10. Add ~50 ml of sterile, ice cold water to the bottle and vortex to resuspend the cell pellet; bring the
volume to 500 ml with sterile, ice-cold water. Pellet the cells by centrifugation at 4000 x g for 5 min
at 4°C; pour off and discard the supernatant.
11. Resuspend the cell pellet in 25–30 ml of sterile, ice-cold 1 M sorbitol and transfer to a chilled 30 ml
Oakridge tube. Pellet the cells by centrifugation at 4000 x g for 5 min at 4°C; pour off and discard
the supernatant.
12. Resuspend the cell pellet in 0.5 ml of sterile, ice-cold 1 M sorbitol; the final cell volume should be
1.3–1.5 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.
7.1.2 Electroporation
1.
For each sample to be electroporated, prepare a 17 x 100 mm sterile tube with 1 ml of 1 M
Sorbitol and place on ice; also, place a 0.2 cm or 0.4 cm cuvette on ice.
2.
Pipette the DNA samples (5–100 ng in a volume of 5 µl) to be electroporated into sterile 1.5 ml
microfuge tubes. Place tubes on ice.
3.
If 0.2 cm cuvettes are used, add 40 µl of the competent cells to each DNA sample; if 0.4 cm
cuvettes are used, add 80 µl of the competent cells to each DNA sample. Mix gently and incubate
on ice for ~5 min.
4.
From the Home screen on Gene Pulser Xcell open the Pre-set Protocols screen, then the Fungal
Protocol screen (press 4, Enter, then 2, Enter). When using 0.2 cm cuvettes, press Enter to open
the S. cerevisiae, 2 mm cuvette Protocol Detail screen. When using 0.4 cm cuvettes, press 2 then
Enter to open the S. cerevisiae, 4 mm Protocol Detail screen. See Section 3.4 for operating
instructions.
5.
Transfer the DNA - cell samples to the appropriate electroporation cuvettes that have been chilled
in ice and tap the suspension to the bottom. Place the cuvette in the ShockPod. Push the lid down
to close. Pulse once.
6.
Remove the cuvette from the chamber, immediately add 1 ml of ice cold 1 M sorbitol to the
cuvette, then gently transfer the diluted cells into a sterile 17 x 100 mm tube.
7.
Check and record the pulse parameters. The time constant should be about 5 milliseconds. The
voltage should be approximately 1.5 kV when pulsing the 0.2 cm cuvettes and approximately 2.5
kV when pulsing the 0.4 cm cuvettes.
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