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

From 2013.igem.org

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       <li>Preparation of the phage (lisis of the donor strain)
       <li>Preparation of the phage (lisis of the donor strain)
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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>Incubation one night at 30°C</li>
                 <li>Incubation one night at 30°C</li>
                 <li>Swab of the phages and bacterias contained in the soft agar</li>
                 <li>Swab of the phages and bacterias contained in the soft agar</li>
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                <li>Add 0,5mL of CHCl3 and 0,5mL of LB for approximately 3 mL of Soft agar</li>
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                <li>Vortex while mixing until obtained a texture type "scratched banana"</li>
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                <li>Centrifugate 10 min at 7000rpm at 4°C</li>
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                <li>Transfer the surnatant in a clean tube and add 0,2mL of CHCl3</li>
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                <li>Stock at 4°C</li>
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      <li>Inoculate of the phage from the diner strain to the recipient strain.
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      <li>Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA.</li>
 
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Revision as of 12:17, 3 October 2013

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Notebook

Protocols

Integration Protocols

Transduction Protocol

  1. 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 10^6 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
  2. 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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