Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 64)

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 B. cereus, press 7 or the Down Arrow to
highlight B. cereus, then press Enter to open the B. cereus Protocol Detail screen. See Section 3.4
for operating instructions.
5. Transfer the DNA - cell samples to the electroporation cuvettes and tap the suspension to the
bottom. Place the cuvette in the ShockPod. Push the chamber lid down to close. Pulse once.
6. Remove the cuvette from the chamber and immediately use a portion of the L broth in the
17 x 100 mm tube to transfer the cells from the cuvette to the tube.
7. Check and record the pulse parameters. The time constant should be about 8.6 msec and the
voltage about 1.0 kV. The field strength can be calculated as actual volts (kV) / cuvette gap (cm).
8. Incubate the cells for 1–1.5 hr at 37°C, shaking at 250 rpm.
9. Plate aliquots of the electroporated cells on LB agar plates containing the appropriate selective
media. Incubate plates overnight at 28°C.
6.4.3 Solutions and Reagents
1. EP buffer (0.5 mM K
2
HPO
4
–KH
2
PO
4
, 0.5 mM MgCl
2
, 272 mM Sucrose): 54.4 ml 1M Sucrose,
100 µl 1 M MgCl
2
, 190 µl 0.1 M KH2PO4, 810 µl 0.1 M K
2
HPO
4
. Bring volume to 200 ml with
Milli-Q water (the pH should be ~7.4). Filter sterilize. Store at 4°C.
2. Brain Heart Infusion (BHI): dissolve 37 g BHI in 1000 ml water; autoclave.
3. L-Broth: 10 g Tryptone peptone, 5 g Yeast extract, 5 g NaCl; dissolve in 1.0 L water. Autoclave.
6.5 Pseudomonas aeruginosa
6.5.1 Preparation of Electrocompetent Cells
From Dennis & Sokol (1995) and Smith & Iglewski (1989).
Using this method, electroporating P. aeruginosa PAO1 with pUCP19, we have obtained transformation
efficiencies >1 x 10
7 transformants/µg.
Gene Pulser Xcell conditions: C = 25 µF; PC = 200 ohm; V = 2.5 kV.
This procedure requires a Gene Pulser Xcell main unit and PC Module.
1. Inoculate a colony of P. aeruginosa into 25 ml of LB in a 125 ml flask; incubate overnight at 37°C
shaking at 300 RPM.
2. Inoculate 10 ml of the overnight culture into a 2.8 L Fernbach flask with 500 ml of LB; incubate at
37°C shaking at 300 RPM.
3. Monitor the culture until OD
600
= 0.5; the cell density should be ~3 x 10 8 cells/ml.
4. Chill the flask on ice for 10 min.
5. Transfer the cells into 2 chilled, sterile 250 ml centrifuge bottles.
6. Pellet the cells by centrifugation at 4°C for 10 min at 2300 x g in a chilled rotor.
7. Discard the supernatant. Resuspend each pellet in 250 ml of sterile, ice cold SMEB buffer. Pellet
the cells as above.
8. Discard the supernatant. Resuspend each pellet in 125 ml of sterile, ice cold SMEB buffer. Pellet
the cells as above.
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