Team:Warsaw/Calendar-Main/12 August 2009

Assembly of endosomal detection operon Marcin Task 1:  Digest of isolate plasmids to verify the success of isolation  Methods:  Samples containing plasmids digested in 11.08.2009 were thawed and loaded into the agarose gel  Comment: Results:   verification of the restriction patterns Comment All isolation were successful Task 2: Isolate the crobox sequence from digested construct Methods: After the digestion reaction mixture was loaded on the gel and electrophoretically separated</li></ul> Results: <img src="http://2009.igem.org/wiki/images/3/3f/Crobox_digest_12_08_09.png" widht="60%" height="60%"> <font face="Times New Roman" size="3"> Elucidation of the digestion Comment: There was no sign of digested crobox sequence. Probably the concentration of the crobox DNA was low. To prepare digested sample it is necessary to use more concentrated solution of the crobox. Task 3:  Transformation of chemocompetent E. coli strain DH5&alpha;</li> </ul> Comment:  Due to obtain more amount previously cloned constructs the bacteria were transformed with followed constructs:  p53 on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li> <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> +<a href="http://partsregistry.org/Part:BBa_C0040"> BBa_C0040</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li> <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> +<a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li> <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> +<a href="http://partsregistry.org/Part:BBa_E0032"> BBa_E0032</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li></ul> Methods:  Detailed protocol of transformation is described <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>.</li> </ul> Task 4:  Restriction digest of biobricks</li> </ul> Comment: Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of previously prepared constructs or biobricks were digested: <a href="http://partsregistry.org/Part:BBa_R0080"> BBa_R0080</a> <a href="http://partsregistry.org/Part:BBa_E0022"> BBa_E0022</a> cro CDS on pKSII vector Methods:  Digest of BBa_R0080 using SpeI and PstI</li> <li>Reaction mixture composition: 20 &mu;l purified plasmid DNA product 0.5 &mu;l SpeI (Fermentas) 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 24 &mu;l MQ water </li></ul> <li>Digest of other constructs using PstI and XbaI</li> <ul><li>Reaction mixture composition: 20 &mu;l purified plasmid DNA product 1 &mu;l XbaI (Fermentas) 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 24 &mu;l MQ water </li></ul> <li>All reaction were performed ~8 hours and both of them were subsequently inactivated via heating in 80&deg;C for 20 minutes</li> <li>The reaction mixtures were loaded on the gel and DNA was electrophoretically separated. In the next step fragments of gel which contain appropriate DNA sequences were cut out and frozen</li><ul>