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How to Make and Transform Electro-Competent Cells

Description of Competent Cells

Competence is a special physiological state of a bacterial cell in which a cell can take up exogenous DNA from the surrounding environment and is not easily decomposed by a restriction endonuclease inside the cell.

Using specific techniques in a laboratory setting, bacterial cells can be transformed into the state of competence for molecular biology applications. For example, the electrical method takes advantage of the low-permeability of bacterial cells in their rapidly-growing phase of their life cycle. By shocking bacteria at this stage with high-intensity transient electric field, the negatively-charged DNA passes through the cell membrane and enters the cell.

DIY Protocol for Generating Electro-Competent Cells

Procedure

• Take frozen cells from freezer and inoculate them on an LB plate at 37°C overnight or for 16-18 hours.
• Once colonies are formed, pick a single colony from the plate and inoculate in a test tube containing 3 ml of LB medium. Incubate overnight at 37°C for 12-16 hours on a shaker (200-300 r/min).
• Add 100ul of the overnight culture into a 250 ml flask containing 50 ml of LB medium and incubate at 37°C about 2-3 hours (200-300 r/min).
• After 2-3 hours when the 600 nm OD value reaches 0.4-0.5, transfer the culture into a cold 50 ml centrifuge tube and incubate on ice for 10-15 minutes. Make sure to maintain the temperature at 4°C and never let the culture warm up again after this step until you are ready for transformation.
• After 10-15 minutes, move the 50 ml centrifuge tube into a pre-cooled (2-4°C) centrifuge machine and centrifuge at 3000-4000 rpm for 10 minutes.
• Discard the supernatant and add 10 ml of ice pre-chilled ddH2O to the centrifuge tube. Gently re-suspend bacterial cells by swirling and proceed with centrifuging at 3000-4000 rpm for 10 minutes.
• Repeat step 6.
• Discard the supernatant and add 1ml of 10% glycerol to re-suspend the bacteria. Transfer cells into 10 Eppendorf tubes and snap-freeze in liquid nitrogen to maintain efficiency.
• Proceed with transformation or store cells at -80°C.

Protocol for Transforming Electro-Competent Cells

Procedure

• Add an appropriate amount of the DNA solution into a vial of competent cells and mix gently.
  • If using frozen competent cell, first thaw them on ice for about 15-20 minutes.
  • Total DNA volume should be less than 10% of the competent cells volume.
  • In general, 10 pg-100 ng DNA is enough for a successful transformation.
• Set the electroporator to a voltage suitable for the bacterial strain you use (usually 1.7-2.5 kv) and pre-chill electroporation cuvettes.
• Transfer the mixture into the pre-chilled cuvettes. Place cuvettes into the electroporation module and press pulse.
• Immediately add 975 µl of 37°C LB/SOC medium and incubate in the 37°C incubator for 1 hour.
• Plate all or part of the culture on one or several LB agar plates containing the appropriate antibiotic.
  • It is best to have at least one plate with 50 µl of culture to avoid overgrowth.
• Incubate plates at 37°C overnight.

Tips from the MolecularCloud Team

• Make sure to turn on the refrigerated centrifuge in advance.
• Pre-cool Eppendorf tubes at -20°C before storing your competent cells to maintain the efficiency of competent cells.
• Make sure to use high-quality glycerin when preparing your stock; glycerin protects cells from damages incurred by ice crystals.
• Avoid frequent freezing and thawing of competent cells as it will reduce the transformation efficiency
• In general, the efficiency of stored competent cells will remains high up to 7 days of storage at -80°C. After that, the efficiency will gradually decrease as the storage time increases.
• Use enough ddH2O to wash the cells; too much ion residue will create strong electrical current and kill the competent cells.