How to make E. coli chemically-competent cells and transform?

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

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 the 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

1. Take frozen cells from freezer and inoculate them on an LB plate at 37°C overnight.

2. 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).

3. 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).

4. 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.

5. 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.

6. 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.

7. Repeat step 6.

8. 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.

9. 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

1. 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.

2. Incubate the mixture on ice for 25~30 minutes.

3. Place the 1/3 bottom of the tube in a 42°C water bath and heat shock for 90~120 seconds.

4. Transfer the tube back on ice and incubate for about 2~3 minutes.

5. Add 0.5~1mL LB medium into the tube and grow cells at 37°C on a shaking incubator for about 45 minutes.

6. 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.

7. Incubate plates at 37°C overnight.

Tips from the MolecularCloud™ Team

1. Pre-cool the 1.5 mL Eppendorf tubes at -20°C before storing your competent cells to maintain the efficiency of competent cells.

2. Make sure to use high-quality glycerin when preparing your stock; glycerin protects cells from damages incurred by ice crystals.

3. Avoid frequent freezing and thawing of competent cells as it will reduce the transformation efficiency.