Team:Warsaw/Calendar-Main/19 July 2009

Assembly of endosomal detection operon Marcin

Task 1:  Digest of  BBa_B0032  Methods:  Digest of BBa_B0032 using SpeI and PstI Reaction mixture composition: 10 &mu;l purified plasmid DNA product 0.5 &mu;l SpeI (Fermentas) 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 34 &mu;l MQ water  The reaction was performed overnight (~12 hours) and it was subsequently inactivated via heating in 80&deg;C for 20 minutes After inactivation sample was electrophoretically separated on the agarose gel:</li> <img src="http://2009.igem.org/wiki/images/c/cc/B0032_digest_18_07_09.png"> Digested product on the gel </ul> Task 2:  Purification of digested product via gel-out</li> </ul> Methods:  Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described here</a></li> Quantification of amount of biobrick DNA after restriction digest:</li> </ul> 1 &mu;l of the digest mixture was diluted to 10 &mu;l and loaded into the gel. Task 3:  Ligations of biobricks</li> </ul> List of designed biobricks: <ol>  BBa_B0032</a>+ BBa_c0040</a> </li>  BBa_B0032</a>+ BBa_C0051</a> </li>  BBa_B0032</a>+<a href="http://partsregistry.org/Part:BBa_E0032"> BBa_E0032</a> </li> </ol> Methods: <ul> <li>Ligation mixture composition: 3 &mu;l digested plasmid with RBS 3 &mu;l digested CDS 5 &mu;l ligation buffer (Fermentas, PEG4000 have been added previously) 1 &mu;l ligase T4 13 &mu;l MQ water </li> <li>Duration of ligation was about 12 hours</li> </ul>

Kama <img src="http://2009.igem.org/wiki/images/9/9d/2009_07_19_ligacja_pho_pKS_opisane.JPG"> <img src="http://2009.igem.org/wiki/images/1/1f/2009_07_19_ligacja2_pho_pKS_opisane.JPG"> Notes <ul> <li>DNA disapeared, probably during inactivation</li> </ul>