Team:Warsaw/Calendar-Main/27 June 2008

Change of the reporter from pZC320 with B-galactosidase to GFP or RFP Piotr, Weronika 

Isolation of pSB1A2 standard plasmids carrying parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator).

Digest of pSB1A2 standard plasmids carrying parts: BBa_E0840(GFP genetrator) and BBa_J04450</a> (RFP generator) with NotI.</li>

 Digest of pZC320</a> with NotI and dephosphorylation</a> with CIAP. </li>

 Gel electrophoresis and gel-out of proper bands. </li>

 Ligation of pZC320</a> with standard parts: <A href=http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840>BBa_E0840</a>(GFP genetrator) and BBa_J04450</a> (RFP generator). </li>

Chemotransformation of E.coli TOP10 with ligation products.</li> Plating transformants on LB+Amp30+X-gal+IPTG.

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Preparation of constructs: OmpA_alpha and OmpA_omega Michał K.  Clean-up</a> of OmpA_alpha and OmpA_omega digest products.

Cloning alpha-A and omega-A fusions on pKS</a> Michał L., Ewa, Marcin <ol> Gel electophoresis of PCL products (Fig. 1</a>).</li> Gel-out</a> of proper bands (alpha-A: 1100 bp and omega-A: 750 bp).</li> <li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Restriction digest</a> of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2B>pKS</a> vector and the PCL products with NotI and SacI (BamHI buffer) for 2 hours.</li> <li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> 1 hour.</li> <li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of Top10 strain and screening on Amp100 plates.</li>

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<img src="http://2008.igem.org/wiki/images/e/ea/PCL_OmegaA_WAW.jpg" width=200/></a> Fig. 1.PCL product - Alpha-A fusion.