Team:Paris/Protocols Competent Bacteria

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Protocol to make competent bacteria

1. Use non competent bacteria (ex: MG1655) stocked in 1.5 mL LB (20% Glycerol): put a sterile tip in the 1.5 mL stock tube and then place it in a 50 mL Falcon with 5 ml LB medium.
Over Night culture at 37°C / 200 rpm

2. 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL

3. Culture at 37°C / 200 rpm untill OD600 reach 0.6

Prepare CaCl2 0.1M.

  • Add 7,351g of CaCl2.2 H2O (FM 147,02) in 500 mL H2O
  • dissolve the CaCl2 by mixing the suspension with the help of a magnetic stirrer
  • Filter the solution with a cell-culture unit of filtration
  • Aliquotes the filtered solution: 25mL in a 50 mL Falcon. Storage at +4°C

4. Fast cooling at +4°C by gently shaking the erlen in ice


5. Use pre-cooled centrifuge at +4°C. Centrifuge 50 mL of the culture in 50 mL falcon: +4°C / 5 min / 5000 rpm

6. Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl2 and mix gently the suspension by up and down

7. Add cold CaCl2 QSP 20 mL and incubate 30 min / +4°C

8. Centrifuge the suspension : +4°C / 5 min / 5000 rpm

9. Discard supernatant by inverting the tube, and resuspend the pellet with 1 mL of cold CaCl2 and mix gently the suspension by up and down

10. Transform or freeze the competent cells. Freeze the competent cells in 50 µL aliquots in the 0.1M CaCl2 medium with 15% glycerol.

11. After transformation, prepare a Glycerol Stock or/and use the transformed bacteria to study the doubling time of the bacteria population

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