Team:Warsaw/Calendar-Main/15 August 2009

Assembly of endosomal detection operon Marcin Task 1: Gel-out of following sequences from the  pSB1A3 plasmid:  p53 CDS  BBa_B0032 + BBa_C0040   BBa_R0080</a>  BBa_E0032</a> </li></ul> </ul></li> </li> </ul> Methods: <ul> Gel-outs were prepared according to manufacturer's manual</li></ul> Results: <img src="http://2009.igem.org/wiki/images/f/f7/Control_gel_out_15_08_09.png" width="60%" heigth="60%"> <font face="Times New Roman" size="3"> Evaluation of gel-outs yield Comment: Some purification were unsuccessful because time required to efficacious activation of the columns is, in fact longer than few minutes (which is not depicted in the manual). It is obligated to repeat digestion and gel-out for the samples which were not isolated Task 2: <ul> Ligation of following constructs:</li> <ul> p53 CDS to  BBa_B0032</a> on  pSB1A3</a> plasmid</li>  BBa_C0040</a> to  BBa_R0080</a> on  pSB1A3</a> plasmid</li> </ul></ul> Methods: <ul> Ligation mix:</li> 1.0 &mu;l - T4 ligase 2.5 &mu;l - Tango buffer (Fermentas, 10x) 3.0 &mu;l - dNTPs (EURx, 5&mu;lM) 6.0 &mu;l - digested vector 12.5 &mu;l - digested insert Ligation time - overnight</li></ul> Task 3: <ul>Prepare the bacterial cultures for isolation of following plasmids:<ul> BBa_E0022</a> with <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li> <li>backbone plasmid <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> </li></ul></ul> Task 4: <ul><li>Transformation of chemocompetent E. coli strain TOP10 to reveal the quality of prepared bacteria</ul></li> Plasmid to transform: <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> Methods: <ul> <li>Detailed protocol of transformation is described <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>.</li> </ul>