Bio-Rad MicroPulser™ Electroporator - Manual (Page 16)

Section 5
High Efficiency Electrotransformation of
E. coli
Electroporation provides a method of transforming E. coli at efficiencies as high as 10 9
to 10 10 transformants/µg, which is greater than is possible with the best chemical methods. The
following protocol describes a method for for preparing and electrotransforming E. coli to
high efficiencies. We are interested in hearing of additional 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.
5.1 Preparation of Electrocompetent Cells
See Ausubel et al. (1987) and Miller and Nickoloff (1995) for additional information.
1. Inoculate 500 ml of L-broth with 1/100 volume of a fresh overnight E. coli culture.
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 therefore depends on the strain and growth conditions).
3. Chill cells on ice for ~20 min. For all subsequent steps, keep the cells as close to 0 °C as
possible (in an ice/water bath) and chill all containers in ice before adding cells. To harvest,
transfer the cells to a cold centrifuge bottle and spin at 4000 x g for 15 minutes at 4 °C.
4. Carefully pour off and discard the supernatant. It is better to sacrifice the 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.
6. Resuspend the pellet in 250 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for
15 minutes at 4 °C; carefully pour off and discard the supernatant.
7. Resuspend the pellet in ~20 ml of ice-cold 10% glycerol. Transfer to a 30 ml sterile
Oakridge tube. Centrifuge at 4000 x g for 15 minutes at 4 °C; carefully pour off and
discard the supernatant.
8. Resuspend the cell pellet in a final volume of 1–2 ml of ice-cold 10% glycerol. The cell
concentration should be about 1–3 x 1010 cells/ml.
This suspension may be frozen in aliquots on dry ice and stored at -70 °C. The cells are
stable for at least 6 months under these conditions.
5.2 Electroporation
1. Thaw the cells on ice. For each sample to be electroporated, place a 1.5 ml microfuge
tube and either a 0.1 or 0.2 cm electroporation cuvette on ice.
2. In a cold, 1.5 ml polypropylene microfuge tube, mix 40 µl of the cell suspension with
1 to 2 µl of DNA (DNA should be in a low ionic strength buffer such as TE). Mix well
and incubate on ice for ~1 minute. (Note: it is best to mix the plasmids and cells in a
microfuge tube since the narrow gap of the cuvettes prevents uniform mixing.)
3. Set the MicroPulser to “Ec1” when using the 0.1 cm cuvettes. Set it to "Ec2" or "Ec3"
when using the 0.2 cm cuvettes. See Section 4 for operating instructions.
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