Team:Warsaw/Calendar-Main/17 June 2008

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<h3>Preparation of constructs with OmpA protein fusions<br>
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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3>
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Michał K.</h3>
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<h4>Michał K.</h4>
<p><ol>
<p><ol>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on OmpA_linker and linker_alpha  with  
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on OmpA_linker and linker_alpha  with  
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  primers (20 cycles, elongation length 1 min 15 s, annealing temperature 57&deg;C).</li>
  primers (20 cycles, elongation length 1 min 15 s, annealing temperature 57&deg;C).</li>
<li> Gel electrophoresis of PCR products and  
<li> Gel electrophoresis of PCR products and  
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<a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of OmpA_alpha and OmpA_omega. </li>
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<a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of OmpA_alpha (1000 bp) and OmpA_omega (800 bp). </li>
<li>Electrophoresis to estimate the concentration of isolated DNA.</li>
<li>Electrophoresis to estimate the concentration of isolated DNA.</li>
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<li> Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of purified OmpA_alpha and OmpA_omega DNA with NdeI and BamHI. </li>
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<li> Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of purified OmpA_alpha and OmpA_omega DNA with NdeI and BamHI (Tango 2x buffer). </li>
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<li> Inoculation of  <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with pACYC177 plasmid into liquid LB + kanamycin. </li>  
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<li> Inoculation of  <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177>pACYC177</a> plasmid into liquid LB + kanamycin. </li>  
</ol></p>
</ol></p>
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<h3> Blue/white and rifampicin test </h3>
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<h3> Blue/white and rifampicin test #2</h3>
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<h4>Michał L., Ewa, Marcin:</h4>
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<h4>Michał L., Ewa</h4>
<p>Repetition of experiment from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/11_June_2008">11/06/2008.</a></p>
<p>Repetition of experiment from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/11_June_2008">11/06/2008.</a></p>
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Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

  1. PCR on OmpA_linker and linker_alpha with OmpaL_N and AlphaP_XB primers (20 cycles, elongation length 1 min 15 s, annealing temperature 57°C).
  2. PCR on OmpA_linker and linker_omega with OmpaL_N and OmegaP_EPB primers (20 cycles, elongation length 1 min 15 s, annealing temperature 57°C).
  3. Gel electrophoresis of PCR products and gel-out of OmpA_alpha (1000 bp) and OmpA_omega (800 bp).
  4. Electrophoresis to estimate the concentration of isolated DNA.
  5. Overnight digest of purified OmpA_alpha and OmpA_omega DNA with NdeI and BamHI (Tango 2x buffer).
  6. Inoculation of E. coli TOP10 with pACYC177 plasmid into liquid LB + kanamycin.

Blue/white and rifampicin test #2

Michał L., Ewa

Repetition of experiment from 11/06/2008.

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