Team:Warsaw/Calendar-Main/21 July 2008

Cloning omega-A fusion on pKS (second attempt) Michał L., Ewa, Marcin We have finally got rid of evil phage infection and we are able to work with E. coli again. Hurray!  Since the phage is gone we can continue this cloning: Restriction digest of PCL product and pKS vector with SacI and NotI (BamHI buffer) . Ligation of omega-A fusion with pKS vector.</li> Transformation</a> of Top10 with ligation product.</li>

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Cloning of protein Z DNA to pET15b-OmpA-omega</a> in place of OmpA Paweł <ol>Result of ligation of pET15b-OmpA-omega </a> with protein Z DNA: 4 colonies grown.</li> Each colony cultured overnight in LB + ampicilin.</li> </ol> Cloning of protein A DNA to OmpA constructs Michał K.  <ol>  Digest</a> of pKS-A</a> plasmid, pACYC177+OmpA_omega</a> and pACYC177+OmpA_alpha</a> with NotI and SacI (BamHI buffer). pACYC177 vectors were also dephosphorylated (with CIAP)</a>.</li>  Gel electrophoresis and gel-out</a> of proper bands (420 bp - <A href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS-A</a> lane, 4050 bp - pACYC177+OmpA_omega</a> lane and 4300 bp pACYC177+OmpA_alpha</a> lane) (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/21_July_2008#fig1">Fig. 1.</a>).</li> <li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of A DNA fragment with both pACYC177 vectors.</li> <li> <a href="http://2008.igem.org/Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of E. coli <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligations. </li> <li>Transformants plating on LB + kanamycin. </li></ol> <img src="http://2008.igem.org/wiki/images/a/ae/Mis_gogo.jpg" width=220/></a> Fig. 1. SacI/NotI digests of plasmids 1. Marker 2. digested pACYC177_OpmA_omega 3. digested pKSA