Team:Paris/Protocols Competent Bacteria

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Protocol to make competent bacteria (iGEM2007)

Prepare CaCl₂ 0.1M

1. Add 7,351g of CaCl₂.2 H₂O (FM 147,02) in 500 mL H₂O
2. dissolve the CaCl₂ by mixing the suspension with the help of a magnetic stirrer
3. Filter the solution with a cell-culture unit of filtration
4. Aliquotes the filtered solution: 25mL in a 50 mL Falcon. Storage at +4°C

Steps

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
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

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

Protocol for competent bacteria by RbCl

Solution for competent bacteria (by RbCl)

• Tampon I (250ml)
  • Potassium acetate (C₂H₃KO₂ 30mM pH 5,8 0,733g
  • RbCl 100mM 3,02g
  • CaCl₂ 10mM 0,3741g
  • MnCl₂50mM 2,5g
  • Glycerol 15% ~37,5ml
  • QS 250ml H₂O ~218,5ml
  • Adjust with Acetic acid (CH₃COOH 100%)to pH 5,8. (Remake the solution if pH beneath 5.8
  • Sterilization (filtration).

• Tampon II (125ml)
  • MOPS 10mM pH 6.5 0,2382g
  • RbCl 10mM 0,150g
  • CaCl₂ 10mM 1,37gg
  • Glycerol 15% ~18,75ml
  • QS 1250ml H₂O ~106.25ml
  • Adjust pH to 6.5 with KOH 1M
  • Sterilization (filtration)

Steps

1. Over night culture (O.N.C)
2. Competent
   1. 50mL of LB with 0.5mL O.N.C
   2. Wait for 5.6 OD₆₀₀
   3. 10mn on ice
   4. centrifuge 10mn at 4000 RPM
   5. resuspend the bacteria pellet in 18mL Tampon I
   6. 10mn on ice
   7. centrifuge 10mn at 4000 RPM
   8. resuspend the bacteria pellet in 8mL Tampon II
   9. Alicot 250µL/tube at -80°C
3. Transformation plasmid (10pg/µl)
   1. 250µl of RbCl competent DH5α
   2. 12,5µl plasmid
   3. Leave 20 min in ice
   4. 45s at 42°C
   5. Leave 2 min in ice
   6. Add 1ml of hot LB (42°C)
   7. 1h at 37 under agitation
   8. Put on plate (LB agar + Antibiotic)
4. culture
   1. take 100µL and spread it on the plate (LB + Antibiotic)
   2. centrifuge 2mn the left
   3. take 100µL and spread it on the plate (LB + Antiobotic)