Team:Paris/Protocols Competent Bacteria

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# 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.<br>Over Night culture at 37°C / 200 rpm
# 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.<br>Over Night culture at 37°C / 200 rpm
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# 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL
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## 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL
# Culture at 37°C / 200 rpm untill OD<sub>600</sub> reach 0.6
# Culture at 37°C / 200 rpm untill OD<sub>600</sub> reach 0.6

Revision as of 19:16, 9 August 2009

Contents

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


  1. . Fast cooling at +4°C by gently shaking the erlen in ice
  1. . Use pre-cooled centrifuge at +4°C. Centrifuge 50 mL of the culture in 50 mL falcon: +4°C / 5 min / 4000 rpm
  1. . 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
  1. . Add cold CaCl2 QSP 20 mL and incubate 30 min / +4°C
  1. . Centrifuge the suspension : +4°C / 5 min / 4000 rpm
  1. . 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
  1. . Transform or freeze the competent cells. Freeze the competent cells in 50 µL aliquots in the 0.1M CaCl2 medium with 15% glycerol.
  1. . 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

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