Team:Warsaw/Calendar-Main/24 July 2008

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<h3>Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA</h3>
 
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<h4>Paweł</h4>
 
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<p><ol>
 
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmids from cultures inocluated on previous day</li>
 
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<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmids with NdeI and NotI. Two positives obtained (proper band: ~150 bp visible)</li>
 
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<li>One of positive plasmids transformed into <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> and plated on LB+amp, overnight, for further isolation of pET15b+Z+omega vector</li>
 
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</ol></p>
 
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<h3>Cloning of protein Z DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b-OmpA-omega</a> in place of OmpA</h3><h4>Paweł</h4>
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<ol><li>Results of ligation: 6 colonies grown.</li>
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<li>Each colony cultured overnight in LB + ampicillin.</li></ol>
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<h3>Cloning of protein A DNA to OmpA constructs</h3><h4>Michał K.</h4>
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<ol>
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<li>Colony PCR on colonies from plates with transformations <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a>. Primers used:
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a>.</li><li> Gel electrophoresis.</li>
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<li>Confirmed transformant colonies inoculated to liquid LB with kanamycin. </li></ol>
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<ol><li>Separate transformant colonies (tranformation of ligation of pACYC177+OmpA_alpha and omega_A from previous day) inoculated to liquid LB with kanamycin.
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<h3>Preparation of chemocompetent bacteria</h3>
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</li></ol>
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<h4>Antoni</h4>
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<h4>Antoni:</h4>
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<ol><li>Preparation of <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemocompetent">chemocompetent</a> bacteria <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htmtop10">TOP10</a>.</li></ol>
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<h3>Cloning omega-A fusion on pKS (second attempt)</h3>
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<h4>Michał L., Ewa, Marcin:</h4>
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<p>Sequencing confirms that we have obtained proper construct with omega-A fusion</p>
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<h3>Cloning of protein A DNA to OmpA constructs</h3>
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<ol><li>Colony PCR on colonies from plates with transformations pACYC177+OmpA_A_omega. Primers used:
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a></li>
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<li>Confirmed transformant colonies inoculated to liquid LB with kanamycin. </li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on previous day (pACYC177+OmpA_A_alpha). </li>
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<li>Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated plasmids with BamHI and SacI.</li></ol>
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</p>
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<h3>Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA<br>
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Paweł</h3>
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<ol><li>Ligations transformed into <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> and plated on LB + ampicillin.</li></ol>
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<p>Preparation of <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemocompetent">chemocompetent</a> bacteria <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htmtop10">TOP10</a>.</p>

Latest revision as of 21:22, 29 October 2008

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Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA

Paweł

  1. Results of ligation: 6 colonies grown.
  2. Each colony cultured overnight in LB + ampicillin.

Cloning of protein A DNA to OmpA constructs

Michał K.

  1. Colony PCR on colonies from plates with transformations pACYC177+OmpA_A_omega. Primers used: AL+SacI and AP+NotI.
  2. Gel electrophoresis.
  3. Confirmed transformant colonies inoculated to liquid LB with kanamycin.

Preparation of chemocompetent bacteria

Antoni

Preparation of chemocompetent bacteria E. coli TOP10.

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