Protocol : Transduction

Bacteriophages stock which packed DNA from Mutant bacteria

1. Mix 5mL of Mutant bacteria with 50µL of CaCl (concentration = 5;10^-3M)

2. Introduce 100µL of this mix in 4 tubes and add increasing concentrations of bacteriophages in each one : 0µL, 10µL, 50µL, 100µL ; wait 20 minutes

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. Introduce the culture in tube and add 0.5mL of CH3Cl

6. Centrifuge at 5000rpm during 10 minutes and collect the supernatant which hold in bacteriophages

Infection

1. Add 100µL of WT bacteria 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 let them 4h at 37°C