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From 2014.igem.org

Transformation of E. coli cells with CaCl₂

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 in -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 100µl 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 0.9ml of LB medium without antibiotic and leave 30min to 1h according to the antibiotic used (for example, 30min for Ampicillin). 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.

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