Team:Warsaw/Calendar-Main/31 August 2009

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  mgtC promoter digest Ligation 

Methods:  The digest mix was prepared as follows: 40&mu;l purified PCR product 5&mu;l Buffer O (Fermentas) 1&mu;l EcoRI enzyme 1&mu;l PstI enzyme 3&mu;l H2O  The digest was carried out in 37&deg;C for 3h and inactivated for 15 min. in 80&deg;C. The product was later purified using Montage PCR Centrifugal Filter Device (Milipore). The ligation mix was prepared as follows: 40&mu;l purified plasmid backbone 20&mu;l purified PCR product 8&mu;l Tango Buffer (Fermentas) 5&mu;l 5mM dNTPs (EURx) 1&mu;l T4 Ligase (Fermentas) 6&mu;l H2O  The ligation was carried out in 18&deg;C overnight and then inactivated in 80&deg;C for 15 min.</li> </ul>

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Ligation to the plasmid</li> </ul>

Methods:  The ligation mix was prepared as follows: 40&mu;l purified plasmid backbone 20&mu;l Cro-Box 8&mu;l Tango Buffer (Fermentas) 5&mu;l 5mM dNTPs (EURx) 1&mu;l T4 Ligase (Fermentas) 6&mu;l H2O </li> The ligation was carried out in 18&deg;C overnight and then inactivated in 80&deg;C for 15 min.</li>

Making of the plac-RBS-llo-intA part Jarek

Tasks:  Preparing the liquid cultures from colonies that grew after transformation and incubationg them in 37C degree.</li>