Team:Warsaw/Calendar-Main/11 August 2009

Assembly of endosomal detection operon Marcin Task 1:  Prepare the bacterial cultures for isolation of plasmid containing  BBa_R0080 and  BBa_E0022  Preparation of bacterial cultures  Prepare LB medium with ampicillin Add 3.5 ml of the medium to the probes Add one bacterial colony to each probe Breed the bacteria about 7 hours </ul> Task 2:  Isolate the plasmid containing BBa_R0080 and BBa_E0022</li> Methods: Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here</a></li></ul> Task 3:  Digest of isolate plasmids verify the success of isolation using EcoRI and PstI</li> </ul> Methods:  Reaction mixture composition: 1 &mu;l purified plasmid DNA product 0.5 &mu;l XbaI (Fermentas) 0.5 &mu;l PstI (Fermentas) 2 &mu;l Buffer Orange (Fermentas) 16 &mu;l MQ water </li> The reaction was performed two 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> Task 4:  Transformation of chemocompetent E. coli strain DH5&alpha;</li> </ul> Methods:  Ligation prepared in 08.08.09 was stopped via thermal inactivation in 80&deg;C for 20 minutes</li> Detailed protocol of transformation is described here</a>.</li> </ul> Task 5: <ul><li>Isolate the crobox sequence from digested construct</li></ul> Methods: <ul><li>After the digestion reaction mixture was loaded on the gel and electrophoretically separated</li></ul> Results: <img src="http://2009.igem.org/wiki/images/6/6d/Digest_11_08_09.png"> <font face="Times New Roman" size="3"> verification of the digestion results Comment: There was no sign of digested crobox sequence. Probably the concentration of the DNA was low or agarose used to prepare the gel was partially degraded. The digestion should be prepare another time. Task 6: <ul> <li>Digest pKS vector to isolate crobox sequence</li> </ul> Methods: <ul> <li>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> <li>The reaction was performed twenty hours and was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li>