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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 OD650nm 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.