Team:Paris/Protocols Competent Bacteria

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Protocol to make competent bacteria (iGEM2007)

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. 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

2nd Protocol for competent bacteria

• Inoculate 50/5 ml of LB with fresh colony or dilution of an overnight culture
• Grow up to OD₆₀₀ 0.5-0.8
• Leave on ice 10min
• Spin 40/2 ml of cell suspension, 5min at 4000rpm, 4°C/minispin 2min at 5000rpm, 4°C
• Resuspend the pellet in 20/1 ml CaCl₂ 50mM (1/2 of initial Vol)
• Leave on ice 10min
• Spin 40/2 ml of resuspension, 5min at 4000rpm, 4°C/minispin 2min at 5000rpm, 4°C
• Resuspend the pellet in 2ml/100µl CaCl₂ 50mM (1/20 of initial Vol)
• Leave on ice overnight before stock at -80°C

Protocol for competent bacteria by RbCl

Solution for competent bacteria (by RbCl)

• Tampon I (250ml)
  • Acétate de potassium 30mM pH 5,8 0,733g
  • RbCl 100mM 3,02g
  • CaCl₂ 10mM 0,3741g
  • MnCl₂50mM 2,5g
  • Glycerol 15% ~37,5ml
  • QSP 250ml H₂O ~218,5ml

• • Ajuster le pH à 5,8 avec de l'acide acétique glacial. Attention si le pH descend en dessous de 5,8 il faut refaire la solution.
  • Stériliser par filtration

• Tampon II (125ml)
  • MOPS 10mM pH 6.5 0,2382g
  • RbCl 10mM 0,150g
  • CaCl₂ 10mM 1,37gg
  • Glycerol 15% ~18,75ml
  • QSP 1250ml H₂O ~106.25ml

• • Ajuster le pH à 6.5 avec du KOH 1M
  • Stérilisation par filtration