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

From 2013.igem.org

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  <h3 class="title3">DeFRT Protocol with FLP recombinase</h2>
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<p class="texte"> 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</p>
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<div class="list">
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      <ol>
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                <li>Transform the strain with the pFLP plasmid</li>
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                <li>Phenotypic expression 2h (selection on Amp) or 3h (on Cm) at 30°C because pFLP is thermo-sensitive</li>
 +
                <li>Spread of the pellet with 100µL of LB on LB agar plate with Amp or Cm</li>
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                <li>Incubation 1 night at 30°C</li>
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                <li>Pick 4 or 5 clones in liquid LB, incubation 2 hours at 30°C then 3 hours at 42°C to express the recombinase</li>
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                <li>Dilute and spread 10^-5 /10^-6 on Lb agar plate.</li>
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                <li>Incubation 1 night at 42°C</li>
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                <li>Right clones are Cm S, Ap S, Kn S</li>
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          </ol>
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</div>
 +
 +
 +
  <h3 class="title3">Integration-excision protocol</h2>
 +
 +
<p class="texte"> 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</p>
 +
 +
 +
<div class="list">
 +
      <ol>
 +
                <li>Transform the strain with the pFLP plasmid</li>
 +
                <li>Phenotypic expression 2h (selection on Amp) or 3h (on Cm) at 30°C because pFLP is thermo-sensitive</li>
 +
                <li>Spread of the pellet with 100µL of LB on LB agar plate with Amp or Cm</li>
 +
                <li>Incubation 1 night at 30°C</li>
 +
                <li>Pick 4 or 5 clones in liquid LB, incubation 2 hours at 30°C then 3 hours at 42°C to express the recombinase</li>
 +
                <li>Dilute and spread 10^-5 /10^-6 on Lb agar plate.</li>
 +
                <li>Incubation 1 night at 42°C</li>
 +
                <li>Right clones are Cm S, Ap S, Kn S</li>
 +
          </ol>
 +
</div>
 +
 +
 +
 +

Revision as of 14:26, 3 October 2013

logo


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



DeFRT Protocol with FLP recombinase

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

  1. Transform the strain with the pFLP plasmid
  2. Phenotypic expression 2h (selection on Amp) or 3h (on Cm) at 30°C because pFLP is thermo-sensitive
  3. Spread of the pellet with 100µL of LB on LB agar plate with Amp or Cm
  4. Incubation 1 night at 30°C
  5. Pick 4 or 5 clones in liquid LB, incubation 2 hours at 30°C then 3 hours at 42°C to express the recombinase
  6. Dilute and spread 10^-5 /10^-6 on Lb agar plate.
  7. Incubation 1 night at 42°C
  8. Right clones are Cm S, Ap S, Kn S

Integration-excision protocol

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

  1. Transform the strain with the pFLP plasmid
  2. Phenotypic expression 2h (selection on Amp) or 3h (on Cm) at 30°C because pFLP is thermo-sensitive
  3. Spread of the pellet with 100µL of LB on LB agar plate with Amp or Cm
  4. Incubation 1 night at 30°C
  5. Pick 4 or 5 clones in liquid LB, incubation 2 hours at 30°C then 3 hours at 42°C to express the recombinase
  6. Dilute and spread 10^-5 /10^-6 on Lb agar plate.
  7. Incubation 1 night at 42°C
  8. Right clones are Cm S, Ap S, Kn S

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