Team:Warsaw/Calendar-Main/2 July 2008

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<h3>Preparation of constructs with OmpA protein fusions
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<h3>Preparation of constructs with OmpA protein fusions<br>
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<br>Paweł</h3>
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Michał K.</h3>
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<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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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP_XB">AlphaP_XB</a>
 
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primers (20 cycles, elongation length 1 min 15 s, annealing temperature 57&deg;C).</li>
 
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on OmpA_linker and linker_omega with
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaP_EPB">OmegaP_EPB</a>
 
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primers (20 cycles, elongation length 1 min 15 s, annealing temperature 57&deg;C).</li>
 
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<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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<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></ol>
 
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<h3>Paweł</h3>
 
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<p><ol><li> Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of pET15b plasmid with NdeI and BamHI, <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation with CIAP</a>.</li></ol></p>
 
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of overnight digest.</li>
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<li> <A HREF="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</A> of OmpA_alfa and pET15b. </li>
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<li> <A HREF="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</A> of OmpA_omega and pET15b.</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htmtop10">TOP10</a> strain with ligation products. </li>
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<li> Transformants plating on LB + ampicillin.</li></ol>
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Preparation of constructs with OmpA protein fusions
Paweł

  1. Clean-up of overnight digest.
  2. Ligation of OmpA_alfa and pET15b.
  3. Ligation of OmpA_omega and pET15b.
  4. Transformation of E. coli TOP10 strain with ligation products.
  5. Transformants plating on LB + ampicillin.