BACTERIAL TRANSFORMATION BY ELECTROPORATION

Bacterial Cel l Preparation:
  1. Inoculate 1 liter of L broth with a 20 ml of an overnight saturated culture of E. coli. Grow to late log phase (A600 = 0.5 - 0.8) while shaking at 37°C. This takes about 3.5 hours.
  2. From this point on, keep cells as close as possible to 0°C.
  3. Chill cells on ice 15 - 30 minutes and centrifuge in a cold rotor 4000 xg for 15 minutes (4500 rpm, in the RC-5B SS-3 rotor ). Discard as much of the supernatant as possible, even if some cells are removed in the process.
  4. Resuspend the pellet in 1 L ice-cold H2O. Centrifuge as above. Resuspend in 0.5 L ice-cold H2O, and centrifuge as above.
  5. Resuspend in 20 ml of ice-cold, sterile 10% glycerol in H2O and transfer to 50 ml disposable Falcon tub e. Centrifuge 3000 rpm, 4°C, 15 minutes. Resuspend in 3 ml of ice-cold sterile 10% glycerol. The cell concentration should be about = 1 - 3 x 1010/ml.
  6. Aliquot 40 µl volumes per microfuge tube. Freeze on dry ice and store at -70°C. Cells are good for about 6 months.

Electroporation (Bio-Rad Gene Pulser):
  1. Thaw bacterial cells at room temperature and then place them on ice. Place sterile cuvettes and the white cuvette chamber slide on ice. Temperature is important because the probability of arcing increases with temperature.
  2. Mix 1-2 µl of DNA (in TE) with the cells. Use 1 pg of supercoiled plasmid DNA as a positive control (this should give about 200 colonies), and 0.1 ng (of vector) for a ligated construct. This may be increased to about 1 ng if low yields are obtained. Incubate on ice for 30-60 seconds.
  3. Set the Gene Pulser apparatus for 25µF. Set the Pulse Controller to 200 O. Set the Gene Pulser to 2.50 kV for 2 mm cuvettes or 1.5 (to 1.8) kV for 1 mm cuvettes. Do not use the 4mm cuvettes for bacteria. E. coli require a high field strength which is proportional to voltage x distance between the electrodes. (For eukaryotic cells, use 0.4 kV, 960µF.)
  4. Transfer the DNA-cell mixture to the bottom of a cold electroporation cuvette. Place in chilled cuvette chamber slide, and push the slide into the chamber to make contact with the electrodes. Pulse once per sample. Remove the cuvette and immediately add 1 ml L broth and resuspend the cells. (A 3-minute delay decreased the transformation efficiency 90%).
  5. Record the time and voltage pulse parameters for each sample. The time constant should be between 4-5 msec. For 1 mm cuvettes, we typically see 3.8 msec with a 1.6 kV setting, 4.0 msec with a 1.7kV setting, and 4.5 msec with a 1.8kV setting.
  6. Transfer the cell suspension to a test tube and incubate 37°C for 1 hour while shaking. Plate cells on agarose with antibiotics, as needed.
  7. The cuvettes may be recycled by soaking them in bleach for a few minutes, washing extensively with tap water, then in double distilled water, and 70% ethanol. Allow to dry. Do not use acetone, and do not autoclave.


RECIPES
L Broth (LB; Luria-Bertani)
10 g tryptone
5 g yeast extract
5 g NaCl
1 L water
Autoclave
TE (10 mM Tris-HCl pH 7.8; 1 mM EDTA)
For 600 ml solution:
0.727 g Tris
1.2 ml of 0.5M EDTA
pH to 7.8 with approximately 7 drops of concentrated HCl with a pasteur pipet (ACID - wear rubber gloves), or about 4 ml of 1 M HCl. QS to 600 ml with water and autoclave.
    Send comments and updates to &nbs p;Dr. Bart Frank, Arthritis and Immunology Program, OMRF
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This protocol was modified from the Bio-Rad Pulse Controller Instruction Manual (#191 90-1062) and is present ed here for convenience.
Accordingly, proper citation for data acquired from this document should be to that manual, as referenced by "Tsugu, H. and Frank, M. B. Bacterial Transformation by Electroporation. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/eporbact.html). 1997. Oklahoma City. Revision Date: October 2, 1997."