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Culture Protocols
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a.menu_sub_active { padding-left: 7px; padding-right: 7px; color:#b0310e; font-weight:bold; }  Main |  Microscope Protocols|  Adapted Protocols|  Culture Protocols|  Molecular biology

 
 * 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

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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

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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

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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
 * 3) 50mL of LB with 0.5mL O.N.C
 * 4) Wait for 5.6 OD600
 * 5) 10mn on ice
 * 6) centrifuge 10mn at 4000 RPM
 * 7) resuspend the bacteria pellet in 18mL Tampon I
 * 8) 10mn on ice
 * 9) centrifuge 10mn at 4000 RPM
 * 10) resuspend the bacteria pellet in 8mL Tampon II
 * 11) Alicot 250&micro;L/tube at -80°C
 * 12) Transformation plasmid (10pg/µl)
 * 13) 250µl of RbCl competent DH5&alpha;
 * 14) 12,5µl plasmid
 * 15) Leave 20 min in ice
 * 16) 45s at 42°C
 * 17) Leave 2 min in ice
 * 18) Add 1ml of hot LB (42°C)
 * 19) 1h at 37 under agitation
 * 20) Put on plate (LB agar + Antibiotic)
 * 21) culture
 * 22) take 100&micro;L and spread it on the plate (LB + Antibiotic)
 * 23) centrifuge 2mn the left
 * 24) take 100&micro;L and spread it on the plate (LB + Antiobotic)

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Glycerol stock

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

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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)