Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 58)

Table 5.1. Resistance of Water in 0.2 cm Cuvettes to which TE has been added 1
SAMPLE R
sample
(ohms) R
sample
(ohms)
(40 µl volume) (200 µl volume)
Water > 600,000 > 600,000
Water + 1 µl TE > 600,000 35,000
Water + 5 µl TE 11,200 8,700
Water + 10 µl TE 4,850 4,700
1 Sample resistance in 0.2 cm cuvettes containing either 40 or 200 µl of water and the indicated volume
of TE (10 mM Tris, pH 8.0, 1 mM EDTA) measured at 1000 V.
Section 6
Electroporation of Bacterial Cells
For most bacterial species, electroporation provides a rapid and simple method for transforming cells.
Particularly for gram negative bacteria, but also for some gram positive species, this method provides
higher transformation efficiencies than conjugation or chemical methods. We are interested in hearing of
additional species and strains transformed by electroporation and including this information in subsequent
versions in our Electroprotocols manual. Please contact your local Bio-Rad representative, access our
web site at
www.bio-rad.com, or, in the U.S., call our Technical Services at (800) 424-6723 with any
comments or questions.
6.1 E. coli
6.1.1 Preparation of Electrocompetent Cells
See Ausubel, et al. (1987) and Miller and Nickoloff (1995) for additional information.
Gene Pulser Xcell conditions: C = 25 µF; PC = 200 ohm; V = 1.8 kV (in 0.1 cm cuvettes);
C = 25 µF; PC = 200 ohm; V = 2.5 kV, or
C = 25 µF; PC = 200 ohm; V = 3.0 kV (in 0.2 cm cuvettes).
This procedure requires a Gene Pulser Xcell main unit and PC Module.
1. Inoculate 5 ml of a fresh overnight E. coli culture into 500 ml of L-broth in a 2.8 L Fernbach flask.
2. Grow the cells at 37°C shaking at 300 rpm to an OD
600
of approximately 0.5–0.7. The best results
are obtained with cells that are harvested at early- to mid-log phase; the appropriate cell density
depends on the strain and growth conditions but should be about 4–5 x 10
7 cells/ml.
3. Chill the cells on ice for ~20 min. For all subsequent steps, keep the cells as close to 0°C as possi-
ble (in an ice/water bath) and chill all containers in ice before adding cells. Transfer the cells to a
sterile, cold 500 ml centrifuge bottle and centrifuge at 4000 x g for 15 minutes at 4°C.
4. Carefully pour off and discard the supernatant. It is better to sacrifice yield by pouring off a few
cells than to leave any supernatant behind.
5. Gently resuspend the pellet in 500 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for
15 minutes at 4°C; carefully pour off and discard the supernatant.
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