Team:Warsaw/Calendar-Main/8 October 2008

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



Preparation of <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a> Piotr  <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:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a>).</li> Control <a href=http://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="http://2008.igem.org/Team:Warsaw/Calendar-Main/8_October_2008#fig1">Fig. 1</a>.</li> </ol>

<img src="http://2008.igem.org/wiki/images/7/7a/Traw_10_10_2008.jpg" width=180></a> 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 <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> Piotr  <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> Control <a href=http://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>

</ol> Preparation of vector for pT7 constructs Michał K. Inoculation of colonies from plate with ligation of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> (with removed XbaI site) to liquid LB + ampicillin.

Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a> Piotr 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.

Preparation of <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a> Michał K.   Colony <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AIDpLinB">AIDpLinB</a>

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="http://2008.igem.org/Team:Warsaw/Calendar-Main/8_October_2008#fig2">Fig. 2</a>. </li>

Inoculation of confirmed colonies to liquid LB + ampicillin. </li></ol>

<img src="http://2008.igem.org/wiki/images/d/d9/Kolonijny_aid_07_10_2008.jpg"></a> Fig. 2.Colony PCR on colonies from plates with transformantions (pSB2K3+AID (BBa_K103001))

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



 <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 ligation <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> (with removed EcoRI site).</li>

 Tranformants plating on LB with tetracycline. </li>

</ol>