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 / 4000 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 / 4000 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. [Team:Paris/Protocols_Transform | Transform] or freeze the competent cells. Freeze the competent cells in 50 µL aliquots in the 0.1M CaCl₂ medium with 15% glycerol.