Team:Warsaw/Calendar-Main/22 July 2009

Assembly of invasion operon Marcin Task 1:  Prepare chemocompetent bacteria Detailed protocol   Incubate 100 ml of liquid bacterial culture until the OD value is 0.55 or little more Incubate the bacteria on the ice for 15 minutes Centrifuge the bacteria at 4&deg;C for 15 minutes Remove the supernatant and add 10 ml of 0.1 M solution of CaCl2 Gently suspend the bacterial pellet and incubate the bacteria on the ice for 45 minutes Centrifuge the bacteria at 4&deg;C for 15 minutes</li> Remove the supernatant and add 2 to 5 ml of 0.1 M solution of CaCl2</li> Prepate the aliquots of suspended bacteria which contain 100 &mu;l of the solution</li> Store the bacteria in -80&deg;C</li> </ol>

Assembly of endosomal detection operon Marcin Task 1:  Transformation of chemocompetent E. coli strain DH5&alpha;</li> </ul> Methods:  Detailed protocol of transformation is described <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>. The only modification is usage of total volume of ligation mixture prepared 20.07.09</li> petri dish were hold in 37&deg;C for 16 hours</li> </ul>

Cloning of p53 coding sequence Marcin Task 1:  Cloning p53 coding sequence to pKS plasmid</li> </ul> Methods:  Ligation mixture composition: 10 &mu;l digested p53 25 &mu;l digested pKS 5 &mu;l ligation buffer (Fermentas) 8 &mu;l MQ water 2 &mu;l ligase T4 </li> Duration of ligation was about 7 hours; reaction was conducted in 16 &deg;c (approximately).</li> </ul> Task 2:  Transformation of chemocompetent E. coli strain DH5&alpha;</li> </ul> Methods:  <li>Ligation reaction was stopped via thermal inactivation in 80&deg;C for 20 minutes</li> <li>Detailed protocol of transformation is described <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>. The only modification is usage of half volume of ligation mixture prepared today</li> </ul>

Isolation of BioBricks from 2008 and 2009 Kit Plates Sebastian Task 1: <ul> <li>Digest of following parts</li> <ul> <li><a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a></li> <li><a href="http://partsregistry.org/Part:BBa_C0040"> BBa_C0040</a></li> <li><a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</a></li> <li><a href="http://partsregistry.org/Part:BBa_E0032"> BBa_E0032</a></li></ul> </ul> Methods: <ul> <li>Digest of BBa_C0040, BBa_C0051 and BBa_E0032 with PstI/XbaI and BBa_B0032 with PstI/SpeI</li> <li>Reaction mixture (BBa_C0040, BBa_C0051 and BBa_E0032): 10ul of plasmid 1ul of each enzyme 5ul of Fermentas Tango Yellow buffer 33ul H2O </li> <li>Reaction mixture (BBa_B0032): 5ul of plasmid 0,5ul of each enzyme 2,5ul of Fermentas Tango Yellow buffer 16ul H2O </li> <li>Time of digest - 6h</li> </ul> Task 2: <ul> <li>Electrophoresis and gel-out</li> </ul> Methods: <ul> <li>Electrophoresis of all samples volume</li> http://2009.igem.org/wiki/images/a/ab/Trawienia22-07.JPG <li>Order of samples: (1,2) BBa_E0032, (3,4) BBa_C0051, (5) generuler DNA Mix, (6,8,9) BBa_C0040, (7) BBa_B0032</li> <li>Gel-out of samples with A&A Biotechnology Kit</li> <li>Electrophoresis of the samples after Gel-out</li> http://2009.igem.org/wiki/images/8/8a/Gel-out.JPG <li>Order of samples: BBa_B0032, BBa_C0040, BBa_C0051, BBa_E0032, generuler DNA mix</li> </ul>

Cloning of the mgtc promoter into the pKSII+ plasmid Kamil

Tasks: <ul> <li>Transformation verification</li> </ul>

Methods: <ul> <li>In order to save on resources colonies that potentially contain the mgtc promoter (the white ones) were marked and the entire dish returned to 37&deg;C for the next 24h.</li> </ul>

</li> </ul>

Construction of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 ">K177012</a>  operon1_part2

Ania Tasks:

<ul> <li>Gel extraction of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032"> BBa_B0032</a>- RBS.3 on the pSB1A2 ampicillin resistant plasmid and gel extracion of <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012"> - lacI repressor</a> </li> <li>Overnight ligation of extracted fragments</li> </ul>