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