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Team:Paris/Protocols Competent Bacteria
From 2009.igem.org
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. 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
