Team:Warsaw/Calendar-Main/3 October 2008

Preparation of linker_alpha (BBa_K103009) Piotr  Transformation of TOP10 with ligation pSB2K3 + linker_alpha (BBa_K103009).  Plating on LB with kanamycin.

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#chemotransform">Transformation</a> of <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligation <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a>.</li>  Plating on LB with kanamycin.</li></ol>

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#chemotransform">Transformation</a> of <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligation <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>  Plating on LB with kanamycin.</li></ol>

Preparation of <a href=http://partsregistry.org/Part:BBa_K103006>OmpA-linker (BBa_K103006)</a> Michał K. <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/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+<a href=http://partsregistry.org/Part:BBa_K103006>OmpA-linker (BBa_K103006)</a>).</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 - proper clones found (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/3_October_2008#fig1">Fig. 1</a>).</li></ol>

<img src="http://2008.igem.org/wiki/images/0/0b/Traw_petxba_omp_07_10_2008_na_3_10.jpg"></a> Fig. 1.Control digest of pSB1A3+OmpA-linker(BBa_K103006) 1. Marker 2-5. EcoRI/PstI control digest pSB1A3 carrying OmpA-linker (BBa_K103006)

Preparation of vector for pT7 constructs Michał K.  <a href="http://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> with XbaI (Tango buffer). DNA ends <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting">blunting</a> with Klenow fragment (3 hr). </li>  Gel electrophoresis and <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band (<a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> (with removed XbaI site) - 6500 bp). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/3_October_2008#fig2">Fig. 2</a>. </li> </ol>

<img src="http://2008.igem.org/wiki/images/c/c9/Go_02_10_2008_na_03_10.jpg"></a> Fig. 2.XbaI digest of pET15b+OmpA_omega 1. Marker 2. XbaI digest pET15b+OmpA_omega