Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 62)

6.3 Agrobacterium tumefaciens
6.3.1 Preparation of Electrocompetent Cells
See Lin (1995) for additional information.
Gene Pulser Xcell conditions: C = 25 µF; PC = 200 ohm; V = 2.4 kV.
This procedure requires a Gene Pulser Xcell main unit and PC Module.
1. Inoculate an aliquot from log phase culture of A. tumefaciens into 1.5 L of YM broth in a 2.8 L
Fernbach flask.
2. Incubate at 30°C overnight, shaking at 300 rpm to a density of 5–10 x 10 7 cells/ml (OD550 ~ 1.0).
3. Decant the cells into sterile 500 ml centrifuge bottles and pellet the cells by centrifugation at 3000 x g
for 10 min at 4°C.
4. Carefully pour off and discard the supernatant; place the centrifuge bottles with the cell pellets on
ice.
5. Add ~50 ml of sterile, ice-cold 10% glycerol to each of the bottles and vortex to resuspend the cell
pellets; bring the volume in each of the centrifuge bottles to 500 ml with sterile, ice-cold 10% glycerol.
Pellet the cells by centrifugation at 3000 x g for 10 min at 4°C; pour off and discard the supernatant.
6. Combine the cell pellets and wash the cells in one bottle as in step 5.
7. Resuspend the cell pellets in 25 ml of sterile, ice-cold 10% glycerol 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.
8. Resuspend the cell pellet in 0.5 ml of sterile, ice-cold 10% glycerol; the final cell volume should be
~1.5 ml and the cell concentration should be ~ 5 x 10
10 cells/ml. The cells may be used immediately
or frozen. To freeze, dispense 200 µl aliquots of the electrocompetent cells in sterile 1.5 ml
microfuge tubes; freeze the cells in an isopropanol-dry ice bath, then store at -70°C. The cells are
stable for about 6 months under these conditions.
6.3.2 Electroporation
1. Pipette the DNA samples (up to 100 ng in no more than 5 µl of TE) to be electroporated into sterile
1.5 ml microfuge tubes. Place tubes on ice.
2. Thaw the electrocompetent A. tumefaciens cells on ice. For each DNA sample to be electroporated,
add 20 µl of electrocompetent cells to each DNA sample; gently tap the tubes to mix.
3. For each DNA sample to be electroporated, add 1 ml of YM broth to a 17 x 100 tube at room
temperature and place a 0.1 cm electroporation cuvette on ice.
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 the Protocol Detail screen for A. tumefaciens,
press 4 or the Down Arrow key to highlight A. tumefaciens, then press Enter. See Section 3.4 for
operating instructions.
5. Transfer the DNA - cell samples to the electroporation cuvettes and tap the suspension to the bot-
tom. 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 the 1 ml of YM 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 5 milliseconds. The
voltage should be about 2.4 kV.
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