Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 66)

6.6 Streptococcus pyogenes
6.6.1 Preparation of Electrocompetent Cells
From Simon & Ferretti (1995).
Using this method, we have obtained transformation efficiencies of 1 x 10 5 transformants/µg
electroporating S.pyogenes CS101 with pDC123 (Chaffin & Rubens, 1998).
Gene Pulser Xcell conditions: C = 25 µF; PC = 200 ohm; V = 2.1 kV.
This procedure requires a Gene Pulser Xcell main unit and PC Module.
1. Inoculate a colony of S. pyogenes from a fresh plate into a 25 ml tube containing 20 ml of THY
with 20 mM Glycine; cap the tube tightly. Incubate overnight at 37°C without shaking.
2. Inoculate 18 ml of the overnight culture into a 500 ml flask containing 250 ml of THY media with 20
mM glycine. Swirl gently to mix and continue incubation (about 2 hrs) until the OD
600
is ~0.2 (the
cell density should be about 3 x 10 7 cells/ml).
3. Chill the culture on ice for 10 min.
4. Transfer the cells into a chilled, sterile 500 ml centrifuge bottle.
5. Pellet the cells by centrifugation at 4°C for 10 min at 6000 x g in a chilled rotor.
6. Pipette off and discard the supernatant. Resuspend the cell pellet in 10 ml of ice-cold sterile Elpo
medium, then bring the volume to ~225 ml. Centrifuge the cells at 8000 x g.
7. Pipette off and discard the supernatant. Resuspend the pellet in 5 ml of ice-cold sterile Elpo medium
and transfer to a 30 ml Oakridge tube. Bring the volume to 30 ml, then centrifuge the cells at
12,000 x g.
8. Pipette off and discard the supernatant. Resuspend the cell pellet in about 1.5 ml of ice-cold sterile
Elpo medium. Keep the cells on ice and use as soon as possible for electroporation.
6.6.2 Electroporation
1. Pipette the DNA samples to be electroporated (0.05–0.5 µg) into sterile 1.5 ml microfuge tubes.
Place tubes on ice. Add 200 µl electrocompetent S. pyogenes cells to each plasmid sample; mix,
and incubate on ice for 5–10 min.
2. Transfer 200 µl of plasmid/cell suspension to chilled 0.2 cm cuvettes on ice; tap the solution to the
bottom of the cuvette.
3. For each sample to be electroporated, prepare a sterile 17 x 100 mm tube containing 1.0 ml of
THY at room temperature.
4. From the Home screen on Gene Pulser Xcell open the Pre-set Protocols screen, then the Bacterial
Protocol screen (press 4, then Enter twice). To select S. pyogenes, press 8 or the Down Arrow to
highlight S. pyogenes, then press Enter to open the S. pyogenes Protocol Detail screen. See
Section 3.4 for operating instructions.
5. Place the cuvette in the ShockPod. Push the chamber lid down to close. Pulse once.
6. Immediately after the pulse place the cuvette on ice for ~ 5 min. Check and record the pulse
parameters. The time constant should be about 9.8 msec and the voltage about 2.1 kV. The field
strength can be calculated as volts (kV) / cuvette gap (cm).
7. Add 1.0 ml of THY broth to the cuvette and transfer the plasmid/cell suspension to the
17 x 100 mm tube. Incubate the cells for 2 hr at 37°C without shaking.
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