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

Bacterial Transformation
Electroporation of E. coli

Electrocompetent Cells and purified plasmid DNA are added to a specialized cuvette with an electrode gap of 1-2 mm, a high-voltage pulse is applied, and a portion of liquid nutrient medium is immediately added to the cells. The cells are then spread onto nutrient Agar containing an antibiotic using a spreader and incubated for 24 hours until well-visible colonies appear.

Ca²⁺-competent E. coli cells can be transformed with an unpurified ligase mixture and virtually any DNA suspended in a moderate-salt buffer. In the case of electrocompetent cells, the DNA must be purified, as high salt concentrations can cause arcing in the electroporator cuvette.

The electric field strength, pulse duration, DNA concentration, and recipient Cell suspension density for any cell culture other than E. coli are determined empirically to achieve a high rate of DNA uptake and ensure cell survival following the electrical breakdown of their Biomembranes.

Since an electroporator is a highly specialized instrument, detailed manufacturer instructions (e.g., from Bio-Rad or Eppendorf) for transforming various cell types are supplied with the device.

Materials and Equipment

Electroporator, electroporator cuvettes (2 mm gap), electrocompetent E. coli XL1-Blue cells, pBluescript II SK+ phagemid DNA solution (0.5 µg/mL), or ligase mixture.

Procedure

1. Precool the cuvette on ice or in a refrigerator for 5-10 min.

2. Thaw the required volume of cells on ice. Vortex gently.

3. Transfer an aliquot of DNA ^a containing 10 pg (2 µL of a 0.5 µg/mL solution diluted 100-fold) into a microcentrifuge tube and add 50 µL of competent cells. Alternatively, add the DNA directly to the cells and mix gently.

4. Mix with a pipet tip; do not pipet up and down vigorously.

5. Place the cuvette into the electroporator chamber.

6. Set the electroporator parameters: 2.5 kV for a 2 mm cuvette gap.

7. Deliver the pulse by pressing the appropriate button. The time constant is typically around 5 ms; this value must be verified.

8. Add 1 mL of SOC medium (Lab 8.2) to the cuvette and pipet up and down 2-3 times.

9. Transfer the electroporated cells to a culture tube and incubate for 0.5-1 h at 37°C ^b with aeration on a shaking incubator.

10. Plate a 50-100 µL aliquot onto 2YT agar supplemented with the appropriate antibiotic.

Notes

^a Salts can be removed from the DNA preparation via microdialysis, or the DNA can be reprecipitated and the pellet thoroughly washed. The easiest way to prevent ligase from entering the cells during transformation with a ligation mixture is to heat-inactivate the enzyme.

^b This incubation period is necessary for the resumption of cellular biochemical processes, the initiation of Antibiotic Resistance Gene METABOLISM/31.html">Transcription, and subsequent Translation.