Team:Warsaw/Calendar-Main/4 August 2009

Cloning of the mgtc promoter into the pSB1A3 plasmid
Monika

Tasks
 * Plasmid assembly

Methods
 * The plasmid digest mix contained: 4&mu;l purified plasmid, 2&mu;l Tango buffer (Fermentas), 0.5&mu;l SpeI enzyme, 0.5&mu;l XbaI enzyme, the solution was topped up with H2O to the final volume of 20&mu;l.
 * The mgtc promoter digest mix contained: 12&mu;l purified promoter, 3&mu;l Tango buffer (Fermentas), 0.5&mu;l SpeI enzyme, 0.5&mu;l XbaI enzyme, the solution was topped up with H2O to the final volume of 30&mu;l.
 * The digest was carried out in 37°C for 3h, but 1&mu;l CIAP enzyme was added 1h before end to mgtc promoter digest mix 1h before the end of incubation. Then enzymes were inactivated in 80°C for 20min.

Assembly of endosomal detection operon Marcin Task 1:  Isolate the plasmid containing  BBa_B0032 and  BBa_C0040 from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis Methods: Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here Task 2:  Digest of isolate plasmids with ligated biobricks to verify the success of ligation   Methods:  Digest of isolate plasmids using XbaI and PstI</li> Reaction mixture composition: 0.5 &mu;l purified plasmid DNA product, 0.5 &mu;l XbaI (Fermentas),0.5 &mu;l PstI (Fermentas), 2 &mu;l Buffer Tango (Fermentas), 16.5 &mu;l MQ water</li></ul> The reaction was performed three hours and it was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li> In the next step reaction mixtures were loaded into the agarose gel to analyse restriction pattern of the plasmids</li></ul> Results: <img src="http://2009.igem.org/wiki/images/8/85/C0040_digest_04_08_09.png" height="35%" width="35%"> <font face="Times New Roman" size="3"> verification of the restriction patterns Comment: All isolated plasmids do not have insert. The cloning must be performed another time Task 3:   Another Cloning of this biobrick</li> </ul> Methods:  Ligation mixture composition: 11 &mu;l both digested fragments, 2.5 &mu;l ligation buffer (Fermentas), 1 &mu;l ligase T4 (Fermentas)</li> Duration of ligation was about 16 hours</li> </ul>

Cloning of p53 coding sequence Marcin

Task 1:  Transformation of chemocompetent E. coli strain DH5&alpha;</li> </ul> Methods:  Detailed protocol of transformation is described here</a>. The only modification is usage of total volume of ligation mixture prepared 03.08.09</li> <li>petri dish will be held in 37&deg;C for 16 hours</li> </ul>