Team:Warsaw/Calendar-Main/3 September 2009

Acquiring the internaline A gene from Listeria monocytogenes st. EDG-e genome
Jarek

Tasks:
 * Digestion of PCR products, separetly with MboI and CaiI restriction endonucleases
 * Electrophoretical segregation of digested samples in 1,5% agarose gel
 * Preparation of another PCR reaction with L.monocytogenes genome DNA

Results:
 * There were no visible products of digestion, even after long treatment with ethidium bromide. Propably the Clean-Up kit isn't working well. I'll try to acquire IntA gene with another PCR.

Preparation of glycerol stocks
 Monika 


 * Preparation of glycerol stocks following the prodcedure described here

pAraC + GFP

plac + RBS + cI + ter

plac + RBS + cI + RBS + GFP + ter

J07037

plac

pSB6A1

Assembly of endosomal detection operon Marcin

Task 1: Methods: Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
 * Isolate the plasmids containing following construct:
 * BBa_K177035 on pSB1A3 plasmid

Task 2: Methods: 20 μl purified plasmid DNA product 1 μl XbaI (Fermentas) 1 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 15 μl MQ water
 * Digestion of plasmids previously described constructs
 * Reaction mixture composition:
 * Digest was performed about six hours and subsequently thermal inactivated

Task 3: Methods:
 * Gel-outs of construct described in Task 2
 * All gel-outs were performed using the EurX gel-out kit according to the manual

Task 4: Methods: 10 μl digested insert 8 μl digested vector 2 μl Tango buffer(Fermentas) 3 μl dNTPs mixture (EurX, concentration 5 mM) 1 μl ligase T4 (Fermentas)
 * Ligation of following constructs:
 * BBa_K177037 on pSB1A3 plasmid
 * cro CDS on pSB1A3
 * Bax CDS on pSB1A3
 * Reaction mixture composition:
 * Reaction were carried out 12 hours and subsequently thermally inactivated.

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  Plasmid isolation Control digest 

Methods:  Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology). The digest mix was prepared as follows: 20&mu;l isolated plasmid 3&mu;l Buffer O (Fermentas) 1&mu;l PstI enzyme 1&mu;l EcoRI enzyme 5&mu;l H2O  The digest was carried out for 3h in 37&deg;C and then inactivated in 80&deg;C for 15min. The results were visualised on 1% agarose gel 

Results:  <img src="http://2009.igem.org/wiki/images/6/65/2009.09.03.jpg"> First four bands (besides the the size ruler) show empty plasmids.</li> </ul> Conclusions:  Respawn and try again in... 3 days.</li> </ul>

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Plasmid isolation</li> Control digest</li> </ul>

Methods:  Plasmids from 3 ml of overnight culture were isolated using the Plasmid Mini kit (A&A Biotechnology).</li> The digest mix was prepared as follows: 20&mu;l isolated plasmid 3&mu;l Buffer O (Fermentas) 1&mu;l PstI enzyme 1&mu;l EcoRI enzyme 5&mu;l H2O </li> The digest was carried out for 3h in 37&deg;C and then inactivated in 80&deg;C for 15min.</li> The results were visualised on 1% agarose gel</li> </ul>

Results:  <img src="http://2009.igem.org/wiki/images/6/65/2009.09.03.jpg"> Final five bands show empty plasmids.</li> </ul> Conclusions:  Respawn and try again in... 3 days.</li> </ul> Cotransformation to obtain cells with following plasmid combinations [<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>][<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012">K177012</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177038">K177038</a>][<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177011">K177011</a>][<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177033">K177033</a>,<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177038">K177038</a>] Ania Tasks: <ul> <li> </li> </ul>