Team:Paris/Protocols Culture
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# Over Night | # Over Night | ||
# Transformation | # Transformation | ||
- | [[team:Paris/Protocols_Culture# | + | #[[team:Paris/Protocols_Culture#Competent bacteria|Competent bacteria]] |
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#[[team:Paris/Protocols_Culture#Glycerol stock|Glycerol stock]] | #[[team:Paris/Protocols_Culture#Glycerol stock|Glycerol stock]] | ||
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Revision as of 20:56, 9 August 2009
Contents |
Protocols
- Over Night
- Transformation
- Competent bacteria
- Glycerol stock
CaCl2 (iGEM Paris2007)
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
CaCl2 (2nd Protocol)
- 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
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
- Over night culture (O.N.C)
- Competent
- 50mL of LB with 0.5mL O.N.C
- Wait for 5.6 OD600
- 10mn on ice
- centrifuge 10mn at 4000 RPM
- resuspend the bacteria pellet in 18mL Tampon I
- 10mn on ice
- centrifuge 10mn at 4000 RPM
- resuspend the bacteria pellet in 8mL Tampon II
- Alicot 250µL/tube at -80°C
- Transformation plasmid (10pg/µl)
- 250µl of RbCl competent DH5α
- 12,5µl plasmid
- Leave 20 min in ice
- 45s at 42°C
- Leave 2 min in ice
- Add 1ml of hot LB (42°C)
- 1h at 37 under agitation
- Put on plate (LB agar + Antibiotic)
- culture
- take 100µL and spread it on the plate (LB + Antibiotic)
- centrifuge 2mn the left
- take 100µL and spread it on the plate (LB + Antiobotic)
Glycerol stock
- 0,66ml of 60% glycerol into cryogenic tube
- 1,32ml of overnight bacterial culture
- Vortex gently
- Stock at -80°C