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Team:INSA Toulouse/contenu/lab practice/notebook/protocols/integration
From 2013.igem.org
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 106 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
DeFRT Protocol with FLP recombinase
- 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 106 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
In order to make a deFRT of a strain, you further need to make competent cells of this strain and have a MINI Prep of the pFLP plasmid
- Transform the strain with the pFLP plasmid
- Phenotypic expression 2h (selection on Amp) or 3h (on Cm) at 30°C because pFLP is thermo-sensitive
- Spread of the pellet with 100µL of LB on LB agar plate with Amp or Cm
- Incubation 1 night at 30°C
- Pick 4 or 5 clones in liquid LB, incubation 2 hours at 30°C then 3 hours at 42°C to express the recombinase
- Dilute and spread 10^-5 /10^-6 on Lb agar plate.
- Incubation 1 night at 42°C
- Right clones are Cm S, Ap S, Kn S
Integration-excision protocol
For integration excision, the strain have to be Streptomycine resistant and you have to make competent cells of this strain.
- Transform the strain with the pMS58 plasmid
- Spread on LB Cm plate 1 night at 30°C
- Streak 4 clones of the previous transformation on LB Cm at 42°C one night
- Streak 4 clones of the first integration on LB Cm at 42°C one night
- Streak 4 clones of the second integration on LB Strp 42°C one night
- Sub-cloned few clones on LB Cm, LB Str and LB Kn at 42°C one night.
Right clone are StR CmS et KnR
