Team:Warsaw/Calendar-Main/26 August 2008

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<h3>Cloning of protein A DNA to pET15b+Omp-alpha plasmid</h3><h4>Antoni</h4>
 
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<p><ol>
 
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<li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on colonies from plates with transformations pGeneart+A Primers used:
 
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI.</a></li>
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<h3>Tests for ampicillin resistance given by protein added to medium</h3><h4>Piotr</h4>
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<li> Lack of confirmed transformant colonies. </li></ol></p>
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<ol><li>
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<p>Purification <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> from antibiotic resistance contamination:<br>
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Inoculation of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>  carrying <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> to liquid LB with kanamycin and with kanamycin and ampicillin.</li>
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<li>Inoculation of all pACYC177+OmpA variants of constructs to LB + 0.25 mM IPTG, kanamycin, ampicillin (50 μg/ml) and 50 μg/ml of protein (Z_alpha and Z_omega; negative control without protein).</li>
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<li>Measurement of growth after 5 hours and on next day.</li></ol>
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<h3>Cloning of protein Z DNA to pET15b+Omp-alpha plasmid</h3><h4>Antoni</h4>
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</p>
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<h3>Cloning of protein A DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA-alpha</a> plasmid</h3><h4>Antoni</h4>
<p><ol>
<p><ol>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> pET15b+OmpA_alpha and pGeneart+Z with NdeI and NotI (Orange buffer), pET15b+OmpA_alpha<a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing"> dephosphorylation with CIAP</a>. </li>
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<li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on colonies from plates with transformations <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> Primers used:
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<li> Gel electrophoresis of digested plasmids and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (200 bp for Z lane and 6200 bp for pET15b lane).  </li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of pET15b+alpha and Z. </li>
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<li> Transformation of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>. </li>
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<li> Transformants plating on LB + ampicillin.</li>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI.</a></li>
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<li> Lack of confirmed transformant colonies. </li></ol></p>
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</ol></p>
 
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<h3>Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega</h3><h4>Michał K.</h4>
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<h3>Cloning of alpha to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a></h3><h4>Michał K.</h4>
<p><ol>
<p><ol>
<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 (both potential constructs). </li>
<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 (both potential constructs). </li>
<li> Control  
<li> Control  
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with FastBamHI and FastSacI (Fast Digest buffer).</li> <li> Gel electrophoresis - still no proper clones founded.</li>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with FastBamHI and FastAcc65I (Fast Digest buffer).</li> <li> Gel electrophoresis - still no proper clones found.</li>
</ol></p>
</ol></p>

Latest revision as of 20:56, 26 October 2008

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Tests for ampicillin resistance given by protein added to medium

Piotr

  1. Purification pACYC177+OmpA_alpha from antibiotic resistance contamination:
    Inoculation of E. coli TOP10 carrying pACYC177+OmpA_alpha to liquid LB with kanamycin and with kanamycin and ampicillin.

  2. Inoculation of all pACYC177+OmpA variants of constructs to LB + 0.25 mM IPTG, kanamycin, ampicillin (50 μg/ml) and 50 μg/ml of protein (Z_alpha and Z_omega; negative control without protein).
  3. Measurement of growth after 5 hours and on next day.

Cloning of protein A DNA to pET15b+OmpA-alpha plasmid

Antoni

  1. Colony PCR on colonies from plates with transformations pGeneart+A Primers used: AP+NotI and AL+SacI.
  2. Lack of confirmed transformant colonies.

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

  1. Isolation of plasmids from cultures inocluated on previous day (both potential constructs).
  2. Control digest of isolated plasmids with FastBamHI and FastAcc65I (Fast Digest buffer).
  3. Gel electrophoresis - still no proper clones found.

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