Team:Warsaw/Calendar-Main/1 October 2009

Isolation of BBa_J63010 from 2009 Kit

Monika

Task:


 * Isolate BioBrick BBa_J63010 from 2009 Kit Plate 1

Methods:
 * Alkaline lysis with A&A Biotechnology Kit, procedure described here


 * Control digestion of BBa_J63010

First reaction mixture composition: 5&mu;l plasmid solution 0,3&mu;l EcoRI (Fermentas) 0,3&mu;l PstI (Fermentas) 2&mu;l Buffer Orange (Fermentas) 12,4 μl MQ water

Second reaction mixture composition: 5&mu;l plasmid solution 0,5&mu;l PvuII (Fermentas) 2&mu;l Buffer Green (Fermentas) 12,3 μl MQ water

Digestion in 37&deg;C for 2,5h

Bistable switch testing

Monika

Tasks:


 * Planting GFP expressing clones on LB medium with tetracycline

Cloning of the cro-box 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 2% agarose gel. 

Results:  <img src="http://2009.igem.org/wiki/images/4/40/2009.10.01.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> Sample 6</li> Sample 7</li> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> Sample 8</li> Sample 9</li> Sample 10</li> Sample 11</li> Sample 12</li> Sample 13</li> <li>Sample 14</li> <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> <li>Sample 15</li> <li>Sample 16</li> <li>Sample 17</li> <li>Sample 18</li> <li>Sample 19</li> <li>Sample 20</li> <li>Sample 21</li> <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> </ol> </ul> Conclusions: <ul> <li>It all looks promising, samples 1, 2, 3, and 7 were sent to be sequenced.</li> </ul>

[UPDATE:] The nucleotides in sample 7 read: "tacctctggcggtgata" (prefix and suffix omitted), which means that we have a proper cro-box!.