Transformation of CaCl₂ competent Escherichia coli cells

E. coli is not naturally competent and there are several ways to prepare competent cells for plasmid DNA transformation. This is the chemical method with CaCl₂.

1. Preparation of competent E. coli cells

Day 1: Incubate one colony from LB plate in LB liquid medium. Shake at 37 °C overnight.

Day 2: Dilute the overnight cell culture 100 to 200 times in 100 ml LB medium. Shake vigorously at 37 °C until OD600nm between 0,2 and 0,3.

Place in an ice bath for few minutes and centrifuge the cells for 10 min at 4,000 rpm at 4 °C.

Resuspend the cell pellet in a half volume of CaCl₂ cold and sterile at 100mM. Leave 5 minutes in ice and centrifuge 10 minutes at 4000rpm at 4°C.

Resuspend the cell pellet in 4ml of CaCl₂ 100mM.

Leave 3 to 4 hours in ice.

Cells remain competent for 24 hours at 4°C (Increase competence after several hours in the cold).

To store at -80°C, add 1ml of glycerol (87%) in 4ml of competent cells.

2. Transformation of competent E.coli cells

Take your competent cells out of -80°C and thaw on ice. Add X µl of DNA (usually 20 ng) in 100 µl of competent cells. Mix gently

Place the competent cell/DNA on ice for 20min.

Incubate cells for 2min 30sec at 42°C. The heat shock is very important

Put the tubes back on ice for 1 to 2 min.

Add 900 μl of LB medium without antibiotic and leave 30min to 1h according to the antibiotic used (for example, 30min for Ampicillin) at 37°C in 151 rpm. This step allows the bacteria to generate the antibiotic resistance proteins.

Then, spread 100 μl onto the petri dishes with LB and appropriate antibiotic.

Grow overnight at 37 °C.