Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 65)

9. Discard the supernatant. Resuspend each pellet in 12 ml of sterile, ice cold SMEB buffer. Transfer
to a chilled 30 ml Oakridge tube.
10. Pellet the cells by centrifugation at 4°C for 10 min at 2300 x g in a chilled rotor.
11. Discard the supernatant. Resuspend the cell pellet in 2.0 ml of SMEB buffer. Keep the cells on ice
and use as soon as possible for electroporation.
6.5.2 Electroporation
1. For each sample to be electroporated, pipette 0.05–0.5 µg of plasmid into a sterile 1.5 ml microfuge
tube. Place tubes on ice. Add 100 µl electrocompetent P. aeruginosa cells to each plasmid sample;
mix, and incubate on ice for 5–10 min.
2. Transfer 100 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 DNA sample to be electroporated, prepare a 17 x 100 tube containing 1.0 ml of SOC 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 P. aeruginosa, press 5 or the Down Arrow to
highlight P. aeruginosa, then press Enter to open the P. aeruginosa 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, use the 1 ml of SOC broth in the 17 x 100 mm tube to transfer the
plasmid/cell suspension from the cuvette to the tube. Check and record the pulse parameters. The
time constant should be about 5 msec and the voltage about 2.5 kV. The field strength can be
calculated as volts (kV) / cuvette gap (cm).
7. Incubate the cells for 1 hr at 37°C shaking at 250–300 RPM.
8. Plate onto LB agar plates with selective antibiotic. Incubate overnight at 37°C.
6.5.3 Solutions and Reagents
1. SMEB buffer (1 mM HEPES, pH 7.0, 1 mM MgCl2, 300 mM Sucrose): 2.0 ml 0.5 M HEPES, pH
7.0, 1.0 ml 1.0 M MgCl
2
, 102.7 g Sucrose. Dissolve in water and bring volume to 1000 ml.
Autoclave. Store at 4°C.
2. L-Broth: 10 g Tryptone peptone, 5 g Yeast extract, 5 g NaCI; dissolve in 1 L water. Autoclave.
3. LB agar plates with selective antibiotic: prepare L broth as above, adding 15 g of agar per liter.
Autoclave. Cool to 55–60°C and add antibiotic. Pour 12–15 ml per 100 mm plate.
4. SOB: 2.0 g Tryptone peptone, 0.5 g Yeast extract, 0.2 ml 5 M NaCl, 0.25 ml 1 M KCl; dissolve in
90 ml water. Adjust pH to 7.0. Bring volume to 100 ml. Autoclave. Add 1.0 ml sterile 1 M MgCl
2
and 1.0 ml sterile 1 M MgSO
4
.
5. SOC: to 100 ml SOB, add 2.0 ml sterile 1 M glucose (sterilize by filtration).
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