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Team:INSA Toulouse/contenu/lab practice/notebook/protocols/integration
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<h1 class="title1">Notebook</h1> | <h1 class="title1">Notebook</h1> | ||
| - | <h2 class="title2">Protocols</h2> | + | <h2 class="title2">Integration Protocols</h2> |
| - | <h3 class="title3 | + | <h3 class="title3">Transduction Protocol</h2> |
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| - | <li>Inoculate of the phage from the | + | <li>Inoculate of the phage from the doner strain to the recipient strain.</li> |
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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>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>Incubation one night at 30°C</li> | ||
| + | <li>Swab of the phages and bacterias contained in the soft agar</li> | ||
| + | <li>Add 0,5mL of CHCl3 and 0,5mL of LB for approximately 3 mL of Soft agar</li> | ||
| + | <li>Vortex while mixing until obtained a texture type "scratched banana"</li> | ||
| + | <li>Centrifugate 10 min at 7000rpm at 4°C</li> | ||
| + | <li>Transfer the surnatant in a clean tube and add 0,2mL of CHCl3</li> | ||
| + | <li>Stock at 4°C</li> | ||
| + | </ol> | ||
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Revision as of 14:06, 3 October 2013
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 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
- 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
- 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
- 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
- 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
- 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
* 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.
