Team:Paris/Protocols Competent Bacteria
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Protocol to make competent bacteria (iGEM2007)
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
Steps
- 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 - 1/100 dilution in LB medium QSP 50 mL in an erlenmeyer of 250 mL
- Culture at 37°C / 200 rpm untill OD600 reach 0.6
- Fast cooling at +4°C by gently shaking the erlen in ice
- Use pre-cooled centrifuge at +4°C. Centrifuge 50 mL of the culture in 50 mL falcon: +4°C / 5 min / 4000 rpm
- 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
- Add cold CaCl2 QSP 20 mL and incubate 30 min / +4°C
- Centrifuge the suspension : +4°C / 5 min / 4000 rpm
- 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
- Transform or freeze the competent cells. Freeze the competent cells in 50 µL aliquots in the 0.1M CaCl2 medium with 15% glycerol.
- 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