Team:Warsaw/Calendar-Main/6 August 2009

Cloning of p53 coding sequence Marcin Task 1:  Isolate the plasmid containing p53 CDS from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis Methods: Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here Results:   Evaluation of the isolation yield Samples denoted as A-C are additional isolations of pSB1A3 plasmid Task 2:  Digest of isolate plasmids with ligated p53 CDS to verify the success of ligation   Methods:  Digest of isolate plasmids using EcoRI and PstI</li> Reaction mixture composition: 0.5 &mu;l purified plasmid DNA product 0.5 &mu;l EcoRI (Fermentas),0.5 &mu;l PstI (Fermentas) 2 &mu;l Buffer Orange (Fermentas) 16.5 &mu;l MQ water </li></ul> The digest was performed three hours and it was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li> In the next step reaction mixtures were loaded into the agarose gel to analyse restriction pattern of the plasmids</li></ul> Results: <img src="http://2009.igem.org/wiki/images/3/3b/P53_digest_06_08_09.png" height="50%" width="50%"> <font face="Times New Roman" size="3"> Verification of the restriction patterns  Comment Four samples showed expected restriction pattern - they contain insert but it has no been determinated whether the orientation of the insert is correct. It is expected to perform another digest using XbaI and PstI to confirm the correctness of ligation Task 2:  Second digest in order to determinate the directiom of insert</li> </ul> Methods:  Digest of isolate plasmids using XbaI and PstI</li> Reaction mixture composition: 0.5 &mu;l purified plasmid DNA product 0.5 &mu;l XbaI (Fermentas) 0.5 &mu;l PstI (Fermentas) 2 &mu;l Buffer Tango (Fermentas) 16.5 &mu;l MQ water </li></ul> The digest was performed three hours and it was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li> In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids</li></ul> Results: <img src="http://2009.igem.org/wiki/images/a/a6/P53%2BpSB_digest_Xba%2BPst_06_08_09.png"> <font face="Times New Roman" size="3"> Verification of the restriction patterns  Comment Only one sample has insert cloned in proper direction - sample nr 3

Assembly of endosomal detection operon Marcin Task 1:  Isolate the plasmid containing  BBa_B0032</a> and  BBa_C0040</a> from bacterial cultures</li> Methods: <li>Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf" here>here</a></li></ul> Task 2: <ul> <li>Digest of isolate plasmids with ligated biobricks to verify the success of ligation using XbaI and PstI<</li> </ul> Methods: <ul> <ul><li> Reaction mixture composition: 0.5 &mu;l purified plasmid DNA product 0.5 &mu;l XbaI (Fermentas) 0.5 &mu;l PstI (Fermentas) 2 &mu;l Buffer Tango (Fermentas) 16.5 &mu;l MQ water </li></ul> <li>The reaction was performed three hours and it was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li> <li>After the inactivation samples were frozen in -20&deg;c</li></ul>