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Protocol

High Efficiency Electro-Transformation of E. coli
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High Efficiency Electro-Transformation of E. coli Electroporation provides a method of transforming E. coli to ef f iciencies greater than those available with the best chemical methods. By subjecting mixtures of cells and DNA to exponentially decaying fields of very high initial amplitude, we routinely obtain 109 to 1010 transformants/ug of DNA with various strains and several plasmids. The survival and transformation of calls is related to the intensity of the f ield (f ield strength--voltage/ distance between electrodes) and the length of the pulse (RC time constant). Several combinations of f ield strength and pulse length that produce extremely high efficiencies of transformation. For example, with pulses of 20 msec we see a steep increase in transformation as we increase the field from 1 to 7 kV/cm, and then a sharp decline with further increases in field strength as cell survival drops. With shorter pulses of 5 msec, transformation rises to a maximum at a field of about 11 kVZcm. In each case transformation efficiency is in the range of low to 1010, depending on the host strain and plasmid, and is reached when 30 to 40% of the cells survive. Thus, we can compensate for shorter pulses by increasing the amplitude of the field. The range of this -compensation is limited, however.
The details of these experiments are presented in reference S. Protocols for preparing and electro-transforming E. coli to high efficiencies are described in Table 2. Table 2: Procedure for High Efficiency Electro-Transformation of E. coli A. Preparation of Cells - Use all sterile tubes and pipettes 1. Inoculate 1 liter of L-broth with 1/100 volume of a fresh overnight culture. 2. Grow cells at 370C with vigorous shaking to an ABS600 of 0.5 to 1 (the best results are obtained with cells that are growing rapidly; the appropriate cell density, therefore, depends on the strain and growth conditions). Normally 4-5 hours. 3. To harvest, chill the flask on ice for 15 to 30 minutes, and centrifuge in a cold rotor at 4,000 xgmay, for 15 min. Everything must be kept ice cold from this point on. 4. Resuspend pellets in a total of 1 liter of a cold, low ionic strength wash medium such as 1 mM hepes, pH 7, or water. Centrifgue as in step 3. 5. Resuspend in 0.5 liter of cold, wash medium. Centrifuge as in step 3. 6. Resuspend in 20 ml 10% glycerol, centrifuge as in step 3. 7. Resuspend to a final volume of 2 to 3 ml in 10% glycerol. The cell concentration should be about 3xlOlO cells/ml. 8. This suspension may be frozen in aliquots on dry ice, and stored at -700C. The cells are good for at least 6 months under these conditions. B. E-lectro-transformation 1. Thaw the cells at room temperature and place them on ice. 2. In a cold, 1.5 ml polypropylene tube, mix 40 ul of the cell suspension with 1 to 2 ul of DNA (DNA should be in a low ionic strength buffer such as TED). Mix well and let sit on ice I minute. 3. Set the Gene Pulser apparatus at 25 uF and 2.5 kV. Set the Pulse Controller to 200 . 4. Transfer the mixture of cells and DNA to a cold, 0.2 cm electroporation cuvette, and shake the suspension to the bottom of the cuvette. Place the cuvette in a chilled safety chamber slide, and push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber. For MC1061/P3 cells I use 800 not 200.
High Efficiency Electro-Transformation of E. coli

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