Team:Warsaw/Calendar-Main/16 June 2008

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<h3>Preparation of constructs with OmpA protein fusions</h3>
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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
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<li>Electrophoresis to estimate the concentration of isolated DNA.</li>
<li>Electrophoresis to estimate the concentration of isolated DNA.</li>
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<h3> Blue/white test </h3>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/a/a3/PCR_Ompa_Alpha_Omega_WAW.jpg" width=300/></a>
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<var><b>Fig. 1.</b> PCR products: linker_alpha (lane 2), linker_omega (lane 3), OmpA_linker (lane 4)</var>
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<h3> Blue/white and rifampicin test </h3>
<h4>Michał L., Ewa</h4>
<h4>Michał L., Ewa</h4>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/a/a3/PCR_Ompa_Alpha_Omega_WAW.jpg" width=300/></a>
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<var><b>Fig. 1.</b> PCR products: linker_alpha (lane 2), linker_omega (lane 3), OmpA_linker (lane 4)</var>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/0/0c/Colony_PCR_pZC_WAW.jpg" width=400/></a>
<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/0/0c/Colony_PCR_pZC_WAW.jpg" width=400/></a>

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Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

  1. PCR on pB30D plasmid with OmpaL_N and OmpaP_link primers (15 cycles, elongation duration 45 s, annealing temperature 63°C).
  2. PCR on pUC19 plasmid with AlphaL_link and AlphaP_XB primers (20 cycles, elongation duration 45 s, annealing temperature 63°C).
  3. PCR on pUC19 plasmid with OmegaL_link and OmegaP_EPB primers (20 cycles, elongation duration 30 s, annealing temperature 58°C).
    As a result we got three PCR products: OmpA_linker and two fragments of TEM1 beta-lactamese: linker_alpha and linker_omega.
  4. Gel electrophoresis of PCR products (Fig. 1) and gel-out of proper bands (OmpA_linker - 500 bp, linker_alpha - 600 bp and linker_omega - 350 bp).
  5. Electrophoresis to estimate the concentration of isolated DNA.

Fig. 1. PCR products: linker_alpha (lane 2), linker_omega (lane 3), OmpA_linker (lane 4)

Blue/white and rifampicin test

Michał L., Ewa

  1. Colony PCR.
    Template: DNA isolated from white colonies
    Primers: pZCseqL and pZCseqR

    Colony PCR program
    TemperatureTimeNo. of cycles
    94°C4:00
    94°C0:3028 cycles
    48°C0:45
    72°C1:30
    72°C10:00
    4°Cinfinite

  2. DNA gel electrophoresis of PCR products. Fig. 2.
  3. Gel-out of proper products (~1200 bp).
  4. Sequencing of proper fragments using primer pZCseqL.


Fig. 2. 1 - DNA ladder; 2 to 11 - colony PCR products (lacZ' gene) from blue and white colonies.