Team:Warsaw/Calendar-Main/15 July 2008

Cloning of protein Z DNA to OmpA constructs Michał K.  Two colonies (pACYC177+OmpA_Z_omega) were inoculated to liquid LB with kanamycin.

Cloning omega-A fusion on pKS (second attempt) Michał L., Ewa, Marcin Polymerase Chain Ligation on linker-A and omega-linker  reisolated PCR product omega-linker - 4 µl reisolated PCR product linker-A - 13.5 µl primer OmegaL+SacI - 2 µl primer AP+NotI - 2 µl</li> Pfu buffer with Mg2+ - 5 µl</li> dNTPs - 1 µl</li> H2o - 22 µl</li> Program:</li> <ol>  95&deg;C - 3'</li>  95&deg;C - 30"</li>  55&deg;C - 45"</li>  68&deg;C - 1'</li>  go to step 2 25 x</li>  68&deg; - 10'  keep in 4&deg;</li> </ol></li> gel electrophoresis of products</li></ul>

Preparation of alpha+A conctruct Antoni <ol>Gradient PCR</a> on alpha+A PCR products with AlphaL+SacI</a> and AP+NotI</a> primers.</li> <li>Gel electrophoresis. Again without satisfying results. <li>Third <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on alpha+A. This time two temperatures of annealing (68&deg;C and 72&deg;C) and gradient of DMSO.</li> <li>Gel electrophoresis (<a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/15_July_2008#fig1">Fig. 1.</a>). <li><a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Gel-out</a> of proper 1000 bp band.</li> </li></ol> <img src="http://2008.igem.org/wiki/images/c/c5/PCRalfa%2BAzDMSO.jpg"></a> Fig. 1.Gradient PCR on alpha+A products. Lanes 1-3, 5, 6 - annealing temperature 68&deg;C and DMSO concentration of 2%, 4%, 6%, 8% and 10% respectively. Lanes 7-11 - 72&deg;C and DMSO concentration increasing in the same maner. Lane 4 and 12 GeneRuler DNA Ladder Mix 1 &mu;g and 0.5 &mu;g respectively.