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

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   <h1 class="title1">Notebook</h1>
   <h1 class="title1">Notebook</h1>
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   <h2 class="title2">Protocols</h2>
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   <h2 class="title2">Integration Protocols</h2>
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   <h3 class="title3">Integration Protocols</h2>
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   <h3 class="title3">Transduction Protocol</h2>
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  <p class="texte"><span class="spantitle">Transduction Protocol</span></p>
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                 <li>Dilution of an overnight culture at 1/100 in LB and CaCL2 5mM</li>
                 <li>Dilution of an overnight culture at 1/100 in LB and CaCL2 5mM</li>
                 <li>Incubation until DO=1</li>
                 <li>Incubation until DO=1</li>
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                 <li>Mix 0,3 mL of bacterias with 10^6 phages P1vir = 10-50µL</li>
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                 <li>Mix 0,3 mL of bacterias with 10<SUP>6</SUP> phages P1vir = 10-50µL</li>
                 <li>Incubation at 37°c during 20 min</li>
                 <li>Incubation at 37°c during 20 min</li>
                 <li>Add 3mL of Soft Agar (50% hot LB + 50% LB agar) with the mix of bacterias then spread on LB agar plate</li>
                 <li>Add 3mL of Soft Agar (50% hot LB + 50% LB agar) with the mix of bacterias then spread on LB agar plate</li>
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<div class="list">
<div class="list">
     <ul class="arrow">
     <ul class="arrow">
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       <li>Inoculate of the phage from the diner strain to the recipient strain.</li>
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       <li>Inoculate of the phage from the doner strain to the recipient strain.</li>
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            <ol>
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                <li></li>
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                <li></li>
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                <li></li>
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                <li></li>
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            </ol>
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     </ul>
     </ul>
</div>
</div>
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<div class="list">
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      <ol>
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                <li>Overnight culture of the recipient strain</li>
 +
                <li>Incubation until DO=1 in LB and CaCl2 5mM</li>
 +
                <li>Mix 500µL of bacterias with phages</li>
 +
                <li>Stir together and vortex quickly</li>
 +
                <li>Incubation 20 min at 37°C</li>
 +
                <li>Centrifugate 4 min at 5000g max</li>
 +
                <li>Wash the pellet with 1 mL of LB with 7,5mM of NaCitrate</li>
 +
                <li>Incubation 1 hour at 37°C</li>
 +
                <li>Centrifugate 4 min at 5000g max</li>
 +
                <li>Wash the pellet with 100 µL of clean LB</li>
 +
                <li>Spread on LB agar plate with NaCitrate 7,5mM and the right selection antibiotic</li>
 +
                <li>Incubation 1 night at 37°C</li>
 +
                <li>Sub cloned on LB agar plate with selection antibiotic and NaCitrate 7,5mM</li>
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          </ol>
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</div>
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<div class="clear"></div>
<div class="clear"></div>
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  <p class="texte"><i>* 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.</i><br>
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Note: <i>There is not enough DNA in each well to perform anything but transformations.</i></p>
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<br><br><br>
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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">
 +
      <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<SUP>-5</SUP> /10<SUP>-6</SUP> on Lb agar plate.</li>
 +
                <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>
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 +
 
 +
  <h3 class="title3">Integration-excision protocol</h2>
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<p class="texte">For integration excision, the strain have to be Streptomycine resistant and you have to make competent cells of this strain.</p>
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 +
 
 +
<div class="list">
 +
      <ol>
 +
                <li>Transform the strain with the pMS58 plasmid</li>
 +
                <li>Spread on LB Cm plate 1 night at 30°C</li>
 +
                <li>Streak 4 clones of the previous transformation on LB Cm at 42°C one night </li>
 +
                <li>Streak 4 clones of the first integration on LB Cm at 42°C one night</li>
 +
                <li>Streak 4 clones of the second integration on LB Strp 42°C one night </li>
 +
                <li>Sub-cloned few clones on LB Cm, LB Str and LB Kn at 42°C one night.<br>Right clone are StR CmS et KnR </li>
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          </ol>
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</div>
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Latest revision as of 17:52, 4 October 2013

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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 106 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

For integration excision, the strain have to be Streptomycine resistant and you have to make competent cells of this strain.

  1. Transform the strain with the pMS58 plasmid
  2. Spread on LB Cm plate 1 night at 30°C
  3. Streak 4 clones of the previous transformation on LB Cm at 42°C one night
  4. Streak 4 clones of the first integration on LB Cm at 42°C one night
  5. Streak 4 clones of the second integration on LB Strp 42°C one night
  6. Sub-cloned few clones on LB Cm, LB Str and LB Kn at 42°C one night.
    Right clone are StR CmS et KnR

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