Molecular Biology. A Practical Guide - Velikov V.A. 2013

Bacterial Transformation
Preparation of Electrocompetent E. coli Cells

Electroporation is an efficient physical method for introducing DNA into bacterial Cells, as well as into cultured plant (protoplast), fungal, animal, and human cells. The method was pioneered by Eberhard Neumann (Neuman, 1982). It has been shown that under optimal conditions, the number of transformants can reach up to 80% of the surviving bacterial cells. While this figure is unattainable with standard chemical transformation, electroporation yields can reach up to 10⁹-10¹⁰ transformants per 1 μg of DNA. The advent of Cell electroporation has significantly advanced bioengineering technologies, which constitute a key priority in The Development of science and technology.

The method is based on the principle that applying a short-term (approx. 5 ms) electrical pulse with a voltage of about 2.5 kV induces transient Pores in the E. coli cell membrane, allowing DNA to enter The Cell.

Although this Procedure is highly efficient, it requires a very pure, desalted DNA preparation. Working with crude preparations either results in electrical arcing or causes a drastic drop in transformation competence.

To prepare “electrocompetent cells,” they are washed repeatedly in a cryoprotectant solution (glycerol) on ice and either flash-frozen in liquid nitrogen or used immediately.

Materials and Equipment

E. coli strain XL1-Blue, glycerol.

Solutions

- YENB medium: 0.75% Yeast extract (Bacto); 0.8% Nutrient Broth, pH 7.5. This medium can be replaced with 2YT or LB medium without any significant reduction in transformation efficiency.

- 10% glycerol.

Procedure

1. Using an inoculation loop, pick several (5–20) bacterial colonies from a plate containing the appropriate selective antibiotic and inoculate them into 500 mL of pre-warmed (37°C) liquid YENB medium in a 2 L flask.

2. Grow the culture for approximately 8 hours in a shaking incubator at 200–300 rpm and 37°C until an optical density of OD₆₀₀⁼0.7 is reached (a range of 0.5–1.0 is acceptable). Chill the culture on ice for 5 minutes at 0°C. All subsequent manipulations must be performed on ice or in a refrigerator at +4°C. Deionized Water and glycerol must be pre-cooled.

3. Centrifuge at 4500 rpm for 10 minutes at +4°C, discard the culture supernatant, centrifuge briefly for 5 seconds at 3000 rpm, and remove any residual medium using a micropipette.

4. Wash the cells twice with 100 mL of cold 10% glycerol. First, resuspend the cells using a micropipette and a vortex mixer. Then, centrifuge for 7–10 minutes at 4500 rpm at +4°C. Discard the supernatant, centrifuge for another 5 seconds at 3000 rpm, and remove any remaining liquid.

5. Wash the cells in 20 mL of 10% glycerol following the same procedure as in step 4.

6. Add 10% glycerol to the pellet in a volume equal to the cell volume (yielding approx. 0.7–2.0 mL of suspension) and resuspend.

7. Aliquot the resulting competent cells. A single electroporation aliquot requires 50 μL, so the total volume should be a multiple of 50. Use immediately or freeze in liquid nitrogen. Frozen cells can be stored for long periods at -70^oC.