Team:Paris/Protocols Culture

From 2009.igem.org

iGEM > Paris > Protocols > Culture Protocols


Contents

Culture Protocols

  1. Over Night
  2. Bacterial transformation
  3. Competent bacteria
  4. Glycerol stock
  5. Ferric citrate growth medium

Bacterial transformation

  • 250µl of DH5α competent bacteria (by RbCl)
  • Add 2µl of DNA (ex Biobrick)
  • Incubation during 20min on ice
  • Heat choc 45second at 42°C
  • Leave on ice 2min
  • Add 1ml of LB preheated at 42°C
  • Incubation 1 hour at 37°C under agitation
  • Put 100µl of culture under plate
  • Centrifuge 2min at 6rpm MAX for 2 min
  • Put 100µl of concentrated culture
  • Incubate overnight at 37°C

Competent bacteria

  1. CaCl2 (iGEM Paris2007)
  2. CaCl2 (2nd Protocol)
  3. RbCl

CaCl2 (iGEM Paris2007)

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

CaCl2 (2nd Protocol)

  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

RbCl

Solutions

  • 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)

Glycerol stock

  1. 0,66ml of 60% glycerol into cryogenic tube
  2. 1,32ml of overnight bacterial culture
  3. Vortex gently
  4. Stock at -80°C

Ferric citrate growth medium

Growth on ferric citrate as the sole iron source was tested on Fec agar plates containing NB medium, 1.5% nutrient agar, 0.2 mM 2,2'-dipyridyl, and 1 mM citrate. (ref Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins. Enz et al, 1999)

Retrieved from "http://2009.igem.org/Team:Paris/Protocols_Culture"