Team:INSA Toulouse/contenu/lab practice/notebook/protocols/integration

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Revision as of 13:50, 3 October 2013

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Notebook

Protocols

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 diner strain to the recipient strain.

* To know which antibiotics to use, look at the plasmid that the part is in. The naming scheme for plasmids is specifically designed to indicate antibiotic resistance.
Note: There is not enough DNA in each well to perform anything but transformations.

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