Team:Warsaw/Calendar-Main/7 October 2008

Preparation of linker_alpha (BBa_K103009) Michał K., Piotr  Isolation of plasmid from culture inoculated on previous day (pSB2K3 + linker_alpha (BBa_K103009)). Control digest of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found. Fig. 1.</li> Inoculation of few more colonies which grown on plate with transformation: pSB2K3</a> + linker_alpha (BBa_K103009)</a> to liquid LB + kanamycin. </li>

<img src="http://2008.igem.org/wiki/images/d/d0/Traw_alfa_omega_07_10_2008.jpg"> Fig. 1.Control EcoRI/PstI digest of pSB2K3+linker_omega (BBa_K103013) 1. and 12. Marker 2-9. Control digests of pSB2K3+linker_alpha (BBa_K103009) 10-17. Control digests of pSB2K3+linker_omega (BBa_K103013)

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Preparation of linker_omega (BBa_K103013)</a> Michał K., Piotr <ol> Isolation</a> of plasmid from culture inoculated on previous day (pSB2K3</a> + linker_omega (BBa_K103013)</a>).</li> Control digest</a> of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found. Fig. 1</a>.</li> Inoculation of few more colonies which grown on plates with transformation: pSB2K3</a> + linker_omega (BBa_K103013)</a> to liquid LB + kanamycin. </li>

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<img src="http://2008.igem.org/wiki/images/d/d0/Traw_alfa_omega_07_10_2008.jpg"></a> Fig. 1.Control EcoRI/PstI digest of pSB2K3+linker_omega (BBa_K103013) 1. and 12. Marker 2-9. Control digests of pSB2K3+linker_alpha (BBa_K103009) 10-17. Control digests of pSB2K3+linker_omega (BBa_K103013)

Preparation of OmpA-linker-omega-linker (BBa_K103016)</a> Michał K., Piotr <ol> <a href=http://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=http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/7_October_2008#fig2">Fig. 2</a>.</li> <li>Inoculation of few more colonies which grown on plate with transformation: <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> to liquid LB + kanamycin. </li></ol>

<img src="http://2008.igem.org/wiki/images/0/0e/Traw_05_10_2008.jpg"></a> Fig. 2. Control digests of pACYC177+OmpA-linker-omega-linker (BBa_K103016) 1. Marker 2.-11. Control digests of pACYC177+OmpA-linker-omega-linker (BBa_K103016) 12. Marker

Preparation of vector for pT7 constructs Piotr <ol><li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with selfligation of <a hrefhttp://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> (with removed XbaI site).</li> <li> Plating on LB with ampicillin.</li></ol>

Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> Piotr <ol><li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligations <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> (without internal EcoRI site).</li> <li> Plating on LB with kanamycin.</li>

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Preparation of <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a> Michał K., Piotr <ol><li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of DNA fragments: <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> (from <a href=http://2008.igem.org/Team:Warsaw/Calendar-Main/1_October_2008>1 October</a>).</li> <li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with above ligation.</li> <li> Plating on LB with ampicillin.</li></ol>

Preparation of <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a> Piotr <ol>

<li><a href="http://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> with EcoRI (EcoRI buffer). DNA ends <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting">blunting</a> with Klenow fragment. </li> <li> Overnight <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> to remove EcoRI site.</li> </ol>