Bio-Rad MicroPulser™ Electroporator - Manual (Page 19)

Section 7
Electroporation of
Agrobacterium tumefaciens
7 .1 Preparation of Electrocompetent Cells
See Lin (1995) for additional information.
1. Inoculate 1.5 L of YM broth in a 2.8 L Fernbach flask with an aliquot from log phase
culture of A. tumefaciens.
2. Incubate at 30°C overnight, shaking at 300 rpm to a density of 5–10 x 10 7 cells/ml.
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. Wash the cells again as in step 5.
7. Resuspend each of the cell pellets in 5 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 1 M sorbitol; the final cell volume
should be ~1.5 ml and the cell concentration should be ~ 5 x 1010 cells/ml. 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.
7.2 Electroporation
1. Pipette the DNA samples (up to 5 µl) to be electroporated into sterile 1.5 ml microfuge
tubes; the DNA should be in either water or TE. Place tubes on ice.
2. 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.
3. 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.
4. Set the MicroPulser to "Agr". See Section 4 for operating instructions.
5. Transfer the DNA—cell samples to the electroporation cuvettes and tap the suspension
to the bottom of the tube. Place the cuvette in the chamber slide. Push the slide into the
chamber until the cuvette is seated between the contacts in the base of the chamber. Pulse
once.
6. Remove the cuvette from the chamber and immediately use the 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 field strength can be calculated as actual volts (kV) / cuvette gap (cm).
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