Team:Warsaw/Calendar-Main/10 July 2008

Preparation of construct pKS with A protein Michał L., Marcin   Inactivation of restriction enzymes and CIAP.  Ligation of digested PCR product and pKS for 2h at room temperature.  Transformation of E. coli TOP10 strain with 7 µl of ligation mix.</li>  Transformants plating on LB + ampicillin + X-gal + IPTG.</li></ol>

Cloning of protein Z DNA to pET15b-OmpA-alpha</a> in place of OmpA Piotr, Antoni <ol>Result of ligation of pET15b-OmpA-alpha </a> with protein Z DNA: 16 colonies grown.</li> Each colony cultured overnight in LB + ampicilin.</li> </ol>

Preparation of constructs: OmpA_alpha and OmpA_omega #2 Michał K. <ol>  Isolation of plasmids</a> from cultures inocluated on previous day. </li>  Control digest</a> of isolated plasmids with BamHI and NotI (BamHI buffer). </li>  Gel electrophoresis (Fig. 1.</a>) - we confirmed pACYC177 + OmpA_omega</a>. We didn't obtain pACYC177 + OmpA_alpha</a> probably because of a mistake in plating. </li>  Ligation</a> of pACYC177</a> and OmpA_alpha (1 hr).</li> <li> Transformation of E. coli <a href="http://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation products: <a href="http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha">pACYC177 and OmpA_alpha</a>.</li> <li> Transformants plating on LB + kanamycin.</li> </ol>

<img src="http://2008.igem.org/wiki/images/9/9c/Trawienie_5_th_july.jpg" width=300 /></a> Fig. 1. Control BamHI and NotI digests of plasmids, which were used in successful colony PCRs  1. Marker 2. slot 1. from yesterday's Omp_A_alpha colony PCR 2. slot 14. from yesterday's Omp_A_alpha colony PCR 2. slot 4. from yesterday's Omp_A_omega colony PCR 2. slot 10. from yesterday's Omp_A_omega colony PCR