Team:Paris Saclay/transduction

From 2013.igem.org

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='''Protocol : Transduction'''=
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='''Protocol '''=
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Bacteriophages stock which packed DNA from Mutant bacteria
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'''P1 bacteriophages stock'''
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1.  Mix 5mL of an overnight culture of E. coli strains (which containts the genetic information that we wish to transduced) with 50µL of CaCl (concentration = 5;10^-3M)
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2.  Dispatch 100µL of the culture in 4 tubes and add a bacteriophages stock in each tube: 0µL (control), 10µL, 50µL, 100µL ; incubate 20 minutes at 37°.
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1. Mix 5mL of Mutant bacteria with 50µL of CaCl (concentration = 5;10^-3M)
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3.   Add 1mL of LB and 3mL of TOP AGAR
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2. Introduce 100µL of this mix in 4 tubs and add increasing concentrations of bacteriophages in each one : 0µL, 10µL, 50µL, 100µL ; wait 20 minutes
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4.   Spread out this mix on LCA plates and let them 6h at 37°C
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3. Add 1mL of LB and 3mL of TOP AGAR
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5.   Recuperate each plate overlay in a tube, add 0.5mL of CH3Cl
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4. Spread out this mix on LCA plates and let them 6h at 37°C
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6.   Centrifuge at 5000rpm during 10 minutes and collect the supernatant which contains the bacteriophage
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5. Introduce the culture in tub and add 0.5mL of CH3Cl
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'''P1 transduction'''
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1.   Add 100µL of the recepient E. coli and 50µL of CaCl (concentration = 5.10^-3M) in 4 Eppendorf tubs
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6. Centrifuge at 5000rpm during 10 minutes and collect the supernatant which hold in bacteriophages
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2.   Add increasing concentrations of bacteriophages : 0µL, 5µL, 10µL, 50µL and wait 20 minutes at 37°C
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3.  Add 1mL of LB + citrate (concentration = 5.10^-3M) and wait 1h at 37°C
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Infection
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4.   Spread out this mix on LB + Antibiotic plates and incubate them overnigh at 37°C
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1. Add 100µL of WT bacteria and 50µL of CaCl (concentration = 5.10^-3M) in 4 Eppendorf tubs
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2. Add increasing concentrations of bacteriophages : 0µL, 5µL, 10µL, 50µL and wait 20 minutes at 37°C
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5.   Antibiotic resistant clone should be present, but not on the control plate.  
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3. Add 1mL of LB+citrate (concentration = 5.10^-3M) and wait 1h at 37°C
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4. Spread out this mix on LB+Antibiotic plates and let them 4h at 37°C
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Latest revision as of 15:06, 4 October 2013

Labwork Overview Biobricks Protocols Safety

Protocol

P1 bacteriophages stock

1. Mix 5mL of an overnight culture of E. coli strains (which containts the genetic information that we wish to transduced) with 50µL of CaCl (concentration = 5;10^-3M)

2. Dispatch 100µL of the culture in 4 tubes and add a bacteriophages stock in each tube: 0µL (control), 10µL, 50µL, 100µL ; incubate 20 minutes at 37°.

3. Add 1mL of LB and 3mL of TOP AGAR

4. Spread out this mix on LCA plates and let them 6h at 37°C

5. Recuperate each plate overlay in a tube, add 0.5mL of CH3Cl

6. Centrifuge at 5000rpm during 10 minutes and collect the supernatant which contains the bacteriophage


P1 transduction

1. Add 100µL of the recepient E. coli and 50µL of CaCl (concentration = 5.10^-3M) in 4 Eppendorf tubs

2. Add increasing concentrations of bacteriophages : 0µL, 5µL, 10µL, 50µL and wait 20 minutes at 37°C

3. Add 1mL of LB + citrate (concentration = 5.10^-3M) and wait 1h at 37°C

4. Spread out this mix on LB + Antibiotic plates and incubate them overnigh at 37°C

5. Antibiotic resistant clone should be present, but not on the control plate.



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