Team:Warsaw/Calendar-Main/22 September 2009

Preparation of biobricks to send Marcin

Task 1: Preparation of bacterial cultures:

Comment:

I prepare liquid bacterial cultures for isolation of followed biobricks:
 * BBa_K177016
 * BBa_K177035
 * BBa_K177036
 * BBa_K177042
 * BBa_K177044
 * BBa_K177046

Task 2: Isolation of plasmid from cultures described in Task 1

Methods:
 * Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here

Assembly of endosome detection operon Marcin

Task 1: DNA digest to create following biobricks:
 * BBa_K177037
 * BBa_K177044

Methods: 15 μl purified plasmid DNA product 1 μl enzyme 1 (Fermentas) 1 μl enzyme 2 (Fermentas) 5 μl Buffer Tango (Fermentas) 28 μl MQ water
 * Constructs to digest:
 * BBa_K177035 - PstI, SpeI
 * BBa_K177035 - PstI, XbaI
 * BBa_K177036 - PstI, SpeI
 * BBa_K177044 - PstI, XbaI
 * Reaction mixture composition:
 * Reactions were carried out 9 hour

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  Plasmid isolation Control digest 

Methods:  The plasmids were isolated from 3&mu;l of overnight liquid cultures using the Plasmid Mini kit (A&A Biotechnology). The digest mix was prepared as follows: 20&mu;l purified plasmid 3&mu;l Orange Buffer (Fermentas) 1&mu;l PstI Enzyme (Fermentas) 1&mu;l EcoRI Enzyme (Fermentas) 5&mu;l H2O  The digest was carried out in 37&deg;C for 4h. The results were visualized on 1% agarose gel. 

Results:  <img src="http://2009.igem.org/wiki/images/2/23/2009.09.22_-_mgtC.jpg"> Gel (from left):</li> <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> Sample 1</li> Sample 2</li> Sample 3</li> Sample 4</li> Sample 5</li> Reference sample</li> Sample 6</li> Sample 7</li> Sample 8</li> Sample 9</li> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> </ol> </ul> Conclusions:  No hits, just a lot of "junk".</li> </ul>

Cloning of the cro-box into the pSB1A3 plasmid Kamil

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

Methods: <ul> <li>The plasmids were isolated from 3&mu;l of overnight liquid cultures using the Plasmid Mini kit (A&A Biotechnology). <li>The digest mix was prepared as follows: 20&mu;l purified plasmid 3&mu;l Orange Buffer (Fermentas) 1&mu;l PstI Enzyme (Fermentas) 1&mu;l EcoRI Enzyme (Fermentas) 5&mu;l H2O </li> <li>The digest was carried out in 37&deg;C for 4h.</li> <li>The results were visualized on 2% agarose gel.</li> </ul>

Results: <ul> <img src="http://2009.igem.org/wiki/images/7/78/2009.09.22_-_cro-box.jpg"> <li>Gel (from left):</li> <ol> <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> <li>Sample 1</li> <li>Sample 2</li> <li>Sample 3</li> <li>Sample 4</li> <li>Sample 5</li> <li>Reference sample</li> <li>Sample 6</li> <li>Sample 7</li> <li>Sample 8</li> <li>Sample 9</li> <li>Sample 10</li> </ol> </ul> Conclusions: <ul> <li>No hits, all plasmids empty.</li> </ul>