Team:Warsaw/Calendar-Main/12 July 2009

Insertion of the pho gene into the pKSII+ plasmid Kama  Plasmid digest mix was prepared as follows: 2&mu;l Tango buffer (Fermentas) 1&mu;l pKSII plasmid 1&mu;l XbaI enzyme 1&mu;l SmaI enzyme the solution was topped up with H2O to the final volume of 20 &mu;l.

Pho gene digest mix was prepared as follows: 2&mu;l Tango buffer (Fermentas) 3&mu;l purified gene 1&mu;l XbaI enzyme the solution was topped up with H2O to the final volume of 20 &mu;l.

The digest was kept for 3h at 37&deg;C, and then the enzymes were inactivated for 20min. at 80&deg;C.

 The ligation mix was prepared as follows: 20&mu;l plasmid 20&mu;l gene 5&mu;l ligation buffer G (Fermentas) 2&mu;l T4 DNA ligase (Fermentas) the solution was topped up with H2O to the final volume of 50 &mu;l. The ligation was carried out in 18&deg;C overnight

(~15h)