Team:Warsaw/Calendar-Main/23 September 2008

MutD5 testing Piotr Inoculation MutD5 carrying: pACYC177+OmpA_Z_alpha, pACYC177+OmpA_Z_omega, pACYC177+OmpA_A_omega and pACYC177+OmpA_omega_&Delta;A to liquid LB + 0.25mM IPTG + kanamycin + 50 μl/ml + prey:Z_alpha for pACYC177+OmpA_Z_omega Z_omega for pACYC177+OmpA_Z_alpha </li> A_apha for pACYC177+OmpA_A_omega</a></li>A_alpha for pACYC177+OmpA_omega_&Delta;A</a></li></ul>

Mutagenesis of protein A Paweł

<ol>PNK phosporylation of mutagenesis products for 30 min at 37&deg;C.</li> PNK heat-inactivated by incubation at 75&deg;C for 15 min.</li> T4 ligase added and incubated for 1 h at room temperature.</li> T4 ligase inactivated by incubation at 55&deg;C for 20 min, then 5U of DpnI added and incubated at 37&deg;C for 3 hours.</li> After DpnI treatment mutagenesis products transformed into TOP10</a> and plated on LB + kanamycin plates.</li></ol>

Preparation of &Delta;A (BBa_K103003)</a> Michał K. <ol>Isolation</a> of plasmid from culture inoculated on previous day.</li> Control digest</a> of isolated plasmid with EcoRI and PstI (Orange buffer). No visible band on 250 bp. Fig. 1</a>.</li> <li> Repettition of <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of isolated DNA fragments of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a> and <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103003>&Delta;A</a> - overnight. </li> </ol>

<img src="http://2008.igem.org/wiki/images/f/f7/Traw_23_09.jpg" width=160/></a> Fig. 1. Control EcoRI/PstIdigest of pSB1A3 1. Marker 2. digested pSB1A3