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How to prepare chemically-competent E. coli and transform them chemically

Description of Competent Cells

Competence is a special physiological state of a bacterial cell in which a cell can take up 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 chemical method takes advantage of the low-permeability of bacterial cells in their rapidly-growing phase of their life cycle. By adding CaCl2 solution to bacteria at low temperatures, bacteria swells into a spherical shape and small holes are created in the cell wall. Under these conditions, foreign DNA molecules can bind to the cell surface of bacteria and form an anti-DNase hydroxy-calcium phosphate complex before being taken up by cells by heat shock.

DIY Protocol for Generating Chemically Competent Cells

Procedure

• Take frozen cells from freezer and inoculate them on an LB plate at 37°C overnight.
• Next day afternoon, 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 100 µl 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).
• 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-cooled 0.1 M CaCl2 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 0.1 M CaCl2 to re-suspend the bacteria. Transfer cells into ten 1.5 ml Eppendorf tubes. To obtain more high-efficiency competent cells, you can add less CaCl2 solution.
• Proceed with transformation or store cells by re-suspending them in a glycerin stock (10% final concentration) and storing at -80°C. Usually, competent cells remain viable for successful transformation up to 2 years of storage.

Protocol for Chemical Transformation of 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.
• Incubate the mixture on ice for 25-30 minutes.
• Place the 1/3 bottom of the tube in a 42°C water bath and heat shock for 90-120 seconds.
• Transfer the tube back on ice and incubate for about 2-3 minutes.
• Add 0.5-1ml LB medium into the tube and grow cells at 37°C on a shaking incubator for about 45 minutes.
• 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

• Pre-cool the 1.5 ml 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.