Team:Warsaw/Calendar-Main/27 August 2009

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  Amplification of the mgtc promoter using new primers Isolation of the pSB1A3 plasmid 

Methods:  The PCR mix was prepared as follows: 5&mu;l buffer B (EURx) 5&mu;l 10mM dNTPs 5&mu;l forward starter 5&mu;l reverse starter 2&mu;l OptiTaq polymerase (EURx) 2&mu;l Salmonella matrix 26 &mu;l H2O  PCR programme: 4min 95&deg;C (30s 95&deg;C, 35s 56&deg;C, 40s 72&deg;C)x28 10min 72&deg;C ~ 4&deg;C   The PCR results were visualised with gel electrophoresis on 1% agarose gel. The plasmid isolation was carried out from 3&mu;l of culture cultivated for 6h in 37&deg;C. The Plasmid Mini kit (A&A Biotechnology) was used.</li> </ul>

Results:  Nothing</li> </ul>

Conclusions:  The temperature was chosen... poorly.</li> </ul>

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Creation of the cro-box double stranded DNA</li> </ul>

Methods:  The mixture of equal volumes (10&mu;l) of both ssDNA was heated to 94&deg;C for 15min. and left to cool.

</ul></ul>

Assembly of endosomal detection operon Marcin

Task 1:  Digest of following constructs:</li> <a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</a> with <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li> <a href="http://partsregistry.org/Part:BBa_E0022"> BBa_E0022</a> with <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li> </ul> </ul> Methods:   Reaction mixture composition:</li> 20 &mu;l purified plasmid DNA product 1 &mu;l XbaI (Fermentas) 1 &mu;l PstI (Fermentas) in the case of C0051 0.7 &mu;l SpeI (Fermentas) in the case of E0022 2 &mu;l Buffer Tango (Fermentas) 15 &mu;l MQ water <li>Digest was performed about seven hours</li> <li>After digestion samples were frozen</li></ul>

Making of the plac-RBS-llo-intA part Jarek

Tasks: <ul> <li>Separation of digested DNA fragments in 0,8% agarose.</li> </ul> Results: <ul> <li>The intA gene that was used might not be the intA gene after all. </li></ul>

Cloning switch 1 regulatory parts [ <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>-PcI.RBS.LacI, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>-PcI.RBS.LacI.PcI.RBS.RFP.terminator, <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>-PLacI.RBS.cI.terminator, <a href="http://partsregistry.org/Part:BBa_K177038">K177038</a>-PLacI.RBS.cI.terminator.PLacI.RBS.GFP.terminator ] into two compatible low copy number plasmids of different antibiotic resistance Ania Tasks: <ul> <li> </li> </ul>