Team:Warsaw/Calendar-Main/25 July 2009

Assembly of endosomal detection operon Marcin Task 1:  Digest of isolate plasmids with ligated biobricks to verify the success of ligation   Methods:  Digest of isolate plasmids using XbaI and PstI 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  The reaction was performed three hours and it was subsequently inactivated via heating in 80&deg;C for 20 minutes. In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids Comment: All isolated plasmids have not insert. The cloning must be performed another time Task 2:  Restriction digest of biobricks</li> </ul> Comment: Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested: <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> <a href="http://partsregistry.org/Part:BBa_C0040"> BBa_c0040</a> <a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</a> <a href="http://partsregistry.org/Part:BBa_E0032"> BBa_E0032</a> Methods:  Digest of BBa_B0032 using SpeI and PstI</li> 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 </li></ul> Digest of other biobricks using PstI and XbaI</li> Reaction mixture composition: 10 &mu;l purified plasmid DNA product 1 &mu;l XbaI (Fermentas) 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 34 &mu;l MQ water </li></ul> Both reaction were performed approximately 7 hours they were subsequently inactivated via heating in 80&deg;C for 20 minutes</li> </ul></li> Task 3:  Ligations of biobricks</li> </ul> Methods:  Ligation mixture composition: 8 &mu;l digested plasmid with RBS 9 &mu;l digested CDS 2 &mu;l ligation buffer (Fermentas, PEG4000 have been added previously) 1 &mu;l ligase T4 (Fermentas) </li> Negative control mixture composition: 8 &mu;l digested plasmid with RBS 2 &mu;l ligation buffer (Fermentas, PEG4000 have been added previously) 1 &mu;l ligase T4 (Fermentas) 9 &mu;l MQ water </li> Duration of ligation was about 12 hours</li> </ul> Construction of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 ">K177012</a>  operon1_part2

Ania Tasks:

<ul> <li>Gel electrophoresis of the obtained clones. </li> <li>Repeat the ligation of the PcI and RBs.3LacI parts.</li></ul> Results: <ul> <li>Only small amount of DNA visible on the gel in 4 samples out of 20. Probably plasmid isolation has been done using too old solution 2. The visible DNA fragments are not the correct clone.</li></ul>