Team:Heidelberg/Notebook/Sensing Group/Notebook/2ndweek

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   Home    Team   Overview    Advisors  </a></li>  Undergraduates  </a></li>  University  </a></li>  DKFZ  </a></li>  BioQuant  </a></li>  BioRegion Rhein-Neckar  </a></li> </ul> </li>  Project</a> <ul class="DropDownMenu" id="MB1-DDM1">  Overall Project  </a></li>  Material & Methods  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Sensing"> Sensing  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Killing_I"> Killing I  - Phages  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Killing_II"> Killing II - Colicin  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Visualization"> Visualization  </a></li> </ul> </li>

<li> <a href="http://2008.igem.org/Team:Heidelberg/Parts" style="color: white">Parts</a> <ul class="DropDownMenu" id="MB1-DDM2"> <li><a href="http://2008.igem.org/Team:Heidelberg/Parts"> Submitted Parts  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Parts/Characterization"> Characterization  </a></li> </ul> </li> <li> <a href="http://2008.igem.org/Team:Heidelberg/Modeling" style="color: white">Modeling</a> <ul class="DropDownMenu" id="MB1-DDM3"> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling"> Overview  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling/Chemotaxis"> Chemotaxis-Colicin  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling/Phage"> Phage Dynamics model  </a></li> </ul> </li> <li> <a href="http://2008.igem.org/Team:Heidelberg/Notebook/Overview" style="color: white">Notebook</a> <ul class="DropDownMenu" id="MB1-DDM5"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"> Sensing >  </a> <ul class="SideMenu" id="MB1-DDM2-SM1"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"> Killing I - Phages >  </a> <ul class="SideMenu" id="MB1-DDM2-SM2"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"> Killing II - Colicin >  </a> <ul class="SideMenu" id="MB1-DDM2-SM3"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/visualization"> Visualization  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/material"> Material & Methods  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/team_meetings"> Team Meetings  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/seminar"> Seminar on Synthetic Biology  </a> </ul> </li> <li style="width: 160px"> <a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview" style="color: white">Human Practice</a> <ul class="DropDownMenu" id="MB1-DDM4"> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview"> Project Overview  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Phips_the_Phage"> Phips the Phage  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Essay"> Essay  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"> Surveys  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"> Open Day  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"> Nobel Prize  </a></li> </ul> </li>

<li> <a href="http://2008.igem.org/Team:Heidelberg/Sponsors" style="color: white">Sponsors</a> </li> </ul>

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Back to the overview

Monday, 08/11/2008

 * preparation of LuxS harboring cells O/N culture

Tuesday, 08/12/2008

 * Miniprep of correctly sequenced LuxS and Glycerol-stock
 * LuxQ no. 3 checked via digestion with NcoI/BamHI (NEBuffer 3 + BSA) --> negative result
 * PCR for LuxQ, LuxQ-1, LuxQ-2, Tar-1, Tar-2 with Taq Mastermix
 * 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
 * preparation of O/N culture for LuxQ no. 3

Wednesday, 08/13/2008

 * PCR-purification of the all PCR-products eluted in 50 µl H2O
 * Miniprep of LuxQ no. 3 O/N culture and digestion with xbaI (NEBuffer 2 + BSA)
 * Fusion-PCR for Fusion-1 and repetition of PCR for LuxQ-2 and LuxQ
 * digestion of pDK48 plasmid and Fusion-1 frament with NcoI/NdeI (0.5 µl) and AgeI (1 µl) in NEBuffer 4

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Thursday, 08/14/2008

 * Gel for LuxQ, LuxQ2
 * sequentiell digestion of Fusion-1 with NcoI and NdeI (NEBuffer 3)
 * Double digestion of pDK48 with NcoI/NdeI (NEBuffer 4)

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Friday, 08/15/2008

 * digestion of Fusion-1 with AgeI in NEBuffer 1
 * gel extraction of sequentially digested Fusion-1 and pDK48 products
 * new digestion of Fusion-1 with NcoI/NdeI (NEBuffer 4) and gel extraction, because previous digestion did not yield expected bands (there should be two small bands at 516bp and 392bp)

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 * Ligation of 5 µl Fusion-1 with 5 µl pDK48 (both digested with NcoI/NdeI )
 * Transformation into DH5α (5 µl Ligation products)

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