Team:Warsaw/Calendar-Main/28 July 2009

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  Plasmid assembly 

Methods:  The plasmid digest mix was prepared as follows: 4&mu;l purified plasmid 2&mu;l Tango buffer (Fermentas) 1&mu;l SpeI enzyme 1&mu;l XbaI enzyme The solution was topped up with H2O to the final volume of 20&mu;l.  The mgtc promoter digest mix was prepared as follows: 10&mu;l purified promoter 2&mu;l Tango buffer (Fermentas) 1&mu;l SpeI enzyme 1&mu;l XbaI enzyme The solution was topped up with H2O to the final volume of 20&mu;l.  The digest was carried out in 37&deg;C for 3h. and then inactivated in 80&deg;C for 20min. The digested plasmid was separated on 1% agarose gel. The plasmid backbone was extracted from agarose gel using the GelOut kit (A&A Biotechnology) accrding to the manufacturers protocol (in the total volume of 50&mu;l). The ligation mix was prepared as follows: 50&mu;l purified backbone 20&mu;l digested promoter 8&mu;l T4 ligase buffer (Fermentas) 2&mu;l T4 ligase (Fermentas) </li> The ligation was carried out in 18&deg;C overnight.</li>

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Construction of RBS.3-listeriolysin

Franek/Ania Tasks:  Transform chemocompetent cells with the ligation mixture </li>