Team:Warsaw/Calendar-Main/23 April 2009

PCR inv Kama

Tasks:  Amplification of inv 

Methods:  PCR mixture's composition: 2,5&mu;l pfu buffer (Fermentas) 2,5&mu;l MgSO4 (Fermentas) 1,5&mu;l primers 1,5&mu;l dNTPs (10 mM) 0,5&mu;l pfu turbo polymerase (od Antka) 1&mu;l template DNA from Yersinia Solution was topped up with H2O to 25&mu;l.    PCR program: inv 4min 95&deg;C (30s 95&deg;C, 35s 58&deg;C, 4min15s 72&deg;C)x3 (30s 95&deg;C, 35s 63&deg;C, 4min15s 72&deg;C)x28 10min 72&deg;C ~ 7&deg;C   Electrophoretic separation on 1% agarose gel 

Results: <img src="http://2009.igem.org/wiki/images/b/b3/2009.04.23_-_PCR_inwazyna_opisany.jpg"/>  Gel (from left)</li> </ul> <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> inv</li> inv (template diluted 10&lowast;)</li> </ol>

No product

Cloning of hly gene into pKSII+ vector Kamil

Tasks:  Purification of the PCR product</li> Plasmid assembly</li> </ul>

Methods:  The purification was carried out using Montage PCR Centrifugal Filter Device P36461 (Milipore) and according to the protocol

supplied.</li> Plasmid digest mix was prepared as follows: 2ul Tango buffer (Fermentas) 1ul pKSII plasmid 1ul XbaI enzyme 1ul SmaI enzyme The solution was topped up with H2O to the final volume of 20 ul.</li> hly gene digest mix was prepared as follows: 2ul Tango buffer (Fermentas) 2ul purified gene 1ul XbaI enzyme the solution was topped up with H2O to the final volume of 20 ul.</li> The digest was kept for 3h at 37&deg;C, and then the enzyme was inactivated for 15min. at 80&deg;C.</li>  The ligation mix was prepared as follows: 1ul plasmid 1ul gene 1ul ligation buffer G (Fermentas) 1ul T4 DNA ligase (Fermentas) The solution was topped up with H2O to the final volume of 10 ul. The ligation was carried out in 18&deg;C overnight (~12h) and the inactivated for 10min. at 65&deg;C.</li>  Electrophoretic separation on 1% agarose gel</li> </ul>

Results: <img src="http://2009.igem.org/wiki/images/f/fa/2009.04.24_-_PCR_inwazyna_%2B_ligacja_opisany.jpg"/>  <li>Gel (from left)</li> </ul> <ol> <li>GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> <li>not relevant</li> <li>not relevant</li> <li>not relevant</li> <li>plasmid after ligation</li> </ol>

The trained eye can distingish between the three isoforms of a plasmid. Looks good enough for transformation.