Team:Warsaw/Calendar-Main/19 August 2009

Assembly of endosomal detection operon Marcin Task 1:  Gel-outs of following constructs digested on 17.08.09: cro CDS on pKSII plasmid  BBa_C0040 with  BBa_B0032 on  pSB1A3 plasmid Methods:  All samples were inactivated via heating in 80 &deg;C for 20 minutes All gel-outs were performed using the EurX gel-out kit according to the manual</a></li></ul> Results: <img src="http://2009.igem.org/wiki/images/e/e1/C0040%2BRBS_cro_19_08_09.png" width="30%" heigth="30%"> <font face="Times New Roman" size="3"> cro digested from pKSII (rigth) Purification of insert assembled with C0040 and RBS failed because of very low DNA concentration after plasmid isolation</ul></li> Task 2:  Ligate cro CDS to  pSB1A3</a> plasmid</li></ul> Methods:  Reaction mixture composition:</li> 10 &mu;l insert 7 &mu;l vector 2.3 &mu;l Buffer Tango (Fermentas) 3 &mu;l dNTPs mixture (EurX) 1 &mu;l T4 ligase (Fermentas) Ligation was performed for about 9 hours and it was subsequently thermally inactivated</li> </ul> Task 3:  Prepare bacterial cultures to isolate following constructs:</li>  BBa_R0080</a> on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid</li> <li><a href="http://partsregistry.org/Part:BBa_C0040"> BBa_C0040</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></ul></ul> Task 4: <ul> <li>Isolate of constructs described in Task 3:</li></ul> Methods: <ul> <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 5: <ul> <li>Digest previously isolated plasmids with C0040+B0032 biobricks:</li></ul> Methods: <ul> <li> Reaction mixture composition:</li> 35 &mu;l purified plasmid DNA product 1 &mu;l XbaI (Fermentas) 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 10 &mu;l MQ water <li>Digest was performed overnight</li> </ul>

Making of the plac-RBS-llo part Jarek

Tasks: <ul> <li>Isolation of plasmid DNA from 20 liquid cultures</li> <li>Digestion of the plasmid DNA with PstI and EcoRI restriction endonucleases</li> </ul>