Team:Warsaw/Calendar-Main/8 October 2008

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<h3>Preparation of <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a></h3>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a></h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
<ol>
<ol>
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<h3>Preparation of <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a></h3>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a></h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
<ol>
<ol>
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmid from culture inoculated on previous day (<a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a>).</li>
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmid from culture inoculated on previous day (<a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a>).</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 EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found.</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 EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/8_October_2008#fig1">Fig. 1</a>.</li>
</ol>
</ol>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_I739204>pACYC177</a> + <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a></h3>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/7a/Traw_10_10_2008.jpg" width=180></a>
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<var><b>Fig. 1.</b>Control EcoRI/PstI digests of pSB2K3+linker_omega (BBa_K103013)<br>
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1. Marker<br>
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2-3. Control EcoRI/PstI digests of pSB2K3+linker_omega (BBa_K103013)<br></var>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a></h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
<ol>
<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 plasmid from culture inoculated on previous day (pACYC177 + OmpA_omega_).</li>
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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 plasmid from culture inoculated on previous day (<a href=http://partsregistry.org/Part:BBa_I739204>pACYC177</a> + <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a>).</li>
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmids with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found.</li>
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmids with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found.</li>
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</ol>
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<h3>Preparation of vector for pT7 constructs</h3>
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<h4>Michał K.</h4>
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<p>Inoculation of colonies from plate with ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> (with removed XbaI site) to liquid LB + ampicillin. </p>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a></h3>
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<h4>Piotr</h4>
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<p> Inoculation of colonies from plate with ligation of <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> (without internal EcoRI site) to liquid LB + kanamycin.</p>
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</ol>
 
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<h3>Preparation of BioBricks</h3>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a></h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
<ol>
<ol>
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<li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpLinB">AIDpLinB</a>
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<li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpLinB">AIDpLinB</a>  
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  primers on colonies from plates with transformations <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+ AID (annealing temperature - 55&deg;C, 60 s of elongation step). </li>
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  primers on colonies from plates with transformations <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+<a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a> (annealing temperature - 55&deg;C, 60 s of elongation step). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/8_October_2008#fig2">Fig. 2</a>. </li>
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<li>Inoculation of confirmed colonies to liquid LB + ampicillin. </li>
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<li>Inoculation of confirmed colonies to liquid LB + ampicillin. </li></ol>
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<li>Inoculation of colonies from plate with ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI to liquid LB + ampicillin. </li>
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</ol>
 
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<h3></h3>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/d/d9/Kolonijny_aid_07_10_2008.jpg"></a>
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<var><b>Fig. 2.</b>Colony PCR on colonies from plates with transformantions (pSB2K3+AID (BBa_K103001))<br></var>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a></h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
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<ol><li> Inoculation of colonies from plate with ligation of <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + pLac_OmpA_omega (without EcoRI site) to liquid LB + kanamycin.</li>
 
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligation <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> without EcoRI site.</li>
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<ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligation <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> (with removed EcoRI site).</li>
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<li> Tranformants planting on LB with tetracycline. </li>
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<li> Tranformants plating on LB with tetracycline. </li>

Latest revision as of 20:22, 28 October 2008

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Preparation of linker_alpha (BBa_K103009)

Piotr

  1. Isolation of plasmid from culture inoculated on previous day (pSB2K3 + linker_alpha (BBa_K103009)).
  2. Control digest of isolated plasmids with EcoRI and PstI (Orange buffer). Gel electrophoresis - still no proper clones found.

Preparation of linker_omega (BBa_K103013)

Piotr

  1. Isolation of plasmid from culture inoculated on previous day (pSB2K3 + linker_omega (BBa_K103013)).
  2. Control digest of isolated plasmids with EcoRI and PstI (Orange buffer). Gel electrophoresis - proper clones found. Fig. 1.
Fig. 1.Control EcoRI/PstI digests of pSB2K3+linker_omega (BBa_K103013)
1. Marker
2-3. Control EcoRI/PstI digests of pSB2K3+linker_omega (BBa_K103013)

Preparation of OmpA-linker-omega-linker (BBa_K103016)

Piotr

  1. Isolation of plasmid from culture inoculated on previous day (pACYC177 + OmpA-linker-omega-linker (BBa_K103016)).
  2. Control digest of isolated plasmids with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found.

Preparation of vector for pT7 constructs

Michał K.

Inoculation of colonies from plate with ligation of pET15b+OmpA_omega (with removed XbaI site) to liquid LB + ampicillin.

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Piotr

Inoculation of colonies from plate with ligation of pSB2K3 + BBa_K103018 (without internal EcoRI site) to liquid LB + kanamycin.

Preparation of AID(BBa_K103001)

Michał K.

  1. Colony PCR with AIDlNrH and AIDpLinB primers on colonies from plates with transformations pSB1A3+AID(BBa_K103001) (annealing temperature - 55°C, 60 s of elongation step). Fig. 2.
  2. Inoculation of confirmed colonies to liquid LB + ampicillin.
Fig. 2.Colony PCR on colonies from plates with transformantions (pSB2K3+AID (BBa_K103001))

Preparation of AID under pBAD/araC (BBa_K103002)

Piotr

  1. Transformation of TOP10 with ligation pMPMT5+AID (with removed EcoRI site).
  2. Tranformants plating on LB with tetracycline.

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