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Team:INSA Toulouse/contenu/lab practice/notebook/protocols/integration
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Notebook
Integration Protocols
Transduction Protocol
- Preparation of the phage (lisis of the donor strain)
- Dilution of an overnight culture at 1/100 in LB and CaCL2 5mM
- Incubation until DO=1
- Mix 0,3 mL of bacterias with 10^6 phages P1vir = 10-50µL
- Incubation at 37°c during 20 min
- Add 3mL of Soft Agar (50% hot LB + 50% LB agar) with the mix of bacterias then spread on LB agar plate
- Incubation one night at 30°C
- Swab of the phages and bacterias contained in the soft agar
- Add 0,5mL of CHCl3 and 0,5mL of LB for approximately 3 mL of Soft agar
- Vortex while mixing until obtained a texture type "scratched banana"
- Centrifugate 10 min at 7000rpm at 4°C
- Transfer the surnatant in a clean tube and add 0,2mL of CHCl3
- Stock at 4°C
- Inoculate of the phage from the doner strain to the recipient strain.
- Overnight culture of the recipient strain
- Incubation until DO=1 in LB and CaCl2 5mM
- Mix 500µL of bacterias with phages
- Stir together and vortex quickly
- Incubation 20 min at 37°C
- Centrifugate 4 min at 5000g max
- Wash the pellet with 1 mL of LB with 7,5mM of NaCitrate
- Incubation 1 hour at 37°C
- Centrifugate 4 min at 5000g max
- Wash the pellet with 100 µL of clean LB
- Spread on LB agar plate with NaCitrate 7,5mM and the right selection antibiotic
- Incubation 1 night at 37°C
- Sub cloned on LB agar plate with selection antibiotic and NaCitrate 7,5mM
- Preparation of the phage (lisis of the donor strain)
- Dilution of an overnight culture at 1/100 in LB and CaCL2 5mM
- Incubation until DO=1
- Mix 0,3 mL of bacterias with 10^6 phages P1vir = 10-50µL
- Incubation at 37°c during 20 min
- Add 3mL of Soft Agar (50% hot LB + 50% LB agar) with the mix of bacterias then spread on LB agar plate
- Incubation one night at 30°C
- Swab of the phages and bacterias contained in the soft agar
- Add 0,5mL of CHCl3 and 0,5mL of LB for approximately 3 mL of Soft agar
- Vortex while mixing until obtained a texture type "scratched banana"
- Centrifugate 10 min at 7000rpm at 4°C
- Transfer the surnatant in a clean tube and add 0,2mL of CHCl3
- Stock at 4°C
- Inoculate of the phage from the doner strain to the recipient strain.
- Overnight culture of the recipient strain
- Incubation until DO=1 in LB and CaCl2 5mM
- Mix 500µL of bacterias with phages
- Stir together and vortex quickly
- Incubation 20 min at 37°C
- Centrifugate 4 min at 5000g max
- Wash the pellet with 1 mL of LB with 7,5mM of NaCitrate
- Incubation 1 hour at 37°C
- Centrifugate 4 min at 5000g max
- Wash the pellet with 100 µL of clean LB
- Spread on LB agar plate with NaCitrate 7,5mM and the right selection antibiotic
- Incubation 1 night at 37°C
- Sub cloned on LB agar plate with selection antibiotic and NaCitrate 7,5mM
