Team:Warsaw/Calendar-Main/2 July 2008

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<h3>Preparation of constructs with OmpA protein fusions</h3>
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<li> PCR on OmpA_linker and linker_alpha  with
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<p>'''Preparation of constructs with OmpA protein fusions'''<br>
 
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1. PCR 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>
<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, annealing length 1.15 min, annealing temperature 57 degrees)<br>
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  primers (20 cycles, annealing length 1 min 15 s, annealing temperature 57&deg;C)</li>
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2. PCR on OmpA_linker and linker_omega with
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<li> PCR 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>
<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, annealing length 1.15 min, annealing temperature 57 degrees)<br>
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  primers (20 cycles, annealing length 1 min 15 s, annealing temperature 57&deg;C)</li>
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3. Gel electrophoresis of PCR products and gel-out of OmpA_alpha and OmpA_omega <br>
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<li> Gel electrophoresis of PCR products and  
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4. Overnight digest of purified OmpA_alpha and OmpA_omega DNA with NdeI and BamHI <br>
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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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5. Overnight digest of pET15b plasmid with NdeI and BamHI, dephosphorylation with CIAP
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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 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>
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Revision as of 18:24, 1 October 2008

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Preparation of constructs with OmpA protein fusions

  1. PCR on OmpA_linker and linker_alpha with OmpaL_N and AlphaP_XB primers (20 cycles, annealing 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, annealing length 1 min 15 s, annealing temperature 57°C)
  3. Gel electrophoresis of PCR products and gel-out of OmpA_alpha and OmpA_omega
  4. Overnight digest of purified OmpA_alpha and OmpA_omega DNA with NdeI and BamHI
  5. Overnight digest of pET15b plasmid with NdeI and BamHI, dephosphorylation with CIAP

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