Team:Warsaw/Calendar-Main/27 July 2009

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  Reamplification of the mgtc promoter 

Methods:  The PCR mix was prepared as follows: 5&mu;l buffer B (EURx) 2&mu;l 5mM dNTPs (EURx) 5&mu;l forward starter 5&mu;l reverse starter 2&mu;l OptiTaq polymerase (EURx) The solution was topped up with H2O to the final volume of 50&mu;l.  PCR programme: 4min 95&deg;C (30s 95&deg;C, 35s 58&deg;C, 40s 72&deg;C)x28 10min 72&deg;C ~ 4&deg;C   The results were visualised with gel electrophoresis on 1% agarose gel. 

Results:  Gel Electrophoresis:</li> </ul> <img src=http://2009.igem.org/wiki/images/3/30/2009.07.27.jpg /> From left: <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> 5&mu;l of PCR mix</li> </ol>

Conclusions:  [Borat]Great success![/Borat] </li> </ul>

Assembly of endosomal detection operon Marcin Task 1:  Prepare the bacterial cultures for isolation of plasmids containing ligated biobricks</li> </ul> Preparation of bacterial cultures  Prepare LB medium with kanamycin</li> Add 3.5 ml of the medium to the probes</li> Add one bacterial colony to each probe</li> Breed the bacteria about 7 hours</li></ul>

Construction of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 ">K177012</a>  operon1_part2

Ania Tasks:  extract plasmid, digest with EcoRI and PstI, gel electrophoresis </li> set up liquid culture from cells transformed with repeated ligation, extract plasmid, digest with EcoRI and PstI, gel electrophoresis.</li></ul> Construction of RBS.3-listeriolysin

Ania Tasks:  <li>Digest vector with RBS.3 with SpeI and PstI </li> <li>Digest llo(wild type) and llo mutated with with XbaI and PstI </li> <li>Extract the correct fragments from the gel</li> <li>Set up a ligation of RBS.3 and wild type llo (since the mutated version turned out not to be correct one)</li> </ul> Results: <img src="http://2009.igem.org/wiki/images/4/43/Ania_rbs3_llowt_llomut.jpg"> Comments: The RBS.3 and wild type listeriolysin are correctly digested, the mutated listeriolysin is not digested, sequencing confirmed that we did not have the correct vesrion of mutated listeriolysin.