Team:Warsaw/Calendar-Main/10 August 2009

Assembly of endosomal detection operon Marcin Comment: Transformation performed 09.08.2009 was unsuccessful. There was only one bacterial colony on the plates. The colony was used to culture the bacteria and isolate plasmids. Unfortunetely the isolated plasmid did not contain the ligated insert. It is necessary to prepare another ligation and transformation. Task 1:  Cloning cro CDS to  pSB1A3 vector  Methods:  Ligation mixture composition: 18 &mu;l digested cro 9 &mu;l digested vector 3 &mu;l ligation buffer (Fermentas) 1 &mu;l ligase T4 (Fermentas)  Duration of ligation was about 15 hours; reaction was conducted in 19 &deg;c (approximately).  Task 2:  Digest pKS vector to isolate crobox sequence  Methods:  Reaction mixture composition: 20 &mu;l purified plasmid solution 0.75 &mu;l XbaI (Fermentas) 0.75 &mu;l PstI (Fermentas) 5 &mu;l Buffer Orange (Fermentas) 23.5 &mu;l MQ water </li> The reaction was performed twenty hours and it was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li> After the inactivation samples were frozen in -20&deg;c</li></ul>