Team:Heidelberg/Notebook/Sensing Group/Notebook/5thweek

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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, 09/01/2008

 * 1.selection for Fusion-1 and Fusion-2 cloning
 * the DNA of the colonies was extracted with MINIprep, and then screen with test digestion: PstI (NEBuffer 3 + BSA)
 * for Fusion-1: if our insert is there: one band of 6101 bp if no inser: two bands: 4388 + 2213 bp
 * for Fusion-2: if our insert is there: one band of 6122 bp if no inser: two bands: 4388 + 2213 bp

PCR of LuxQ and two fusion protein constructs
 * Lyse V. harveyi for 5 min @ 99 °C
 * PCR with Phusion (template: supernatend of boiled V. harveyi (a), colony (b))
 * 5 min @ 98 °C || 20s @ 98 °C | 40s @ 58 °C | 1min @ 72 °C || 10 min @ 72 °C | 4 °C hold ( 35 cycles )
 * 35(34) µl H2O, 10 µl 5x HF, 0.5 µl each primer, 1µl Template, 2µl dNTPs, 1µl Phusion, 0(1) µl DMSO
 * PCR with Pfu
 * 3 min @ 94 °C || 45s @ 94 °C | 45s @ 58 °C | 2min @ 72 °C || 10 min @ 72 °C | 4 °C hold ( 35 cycles )
 * 40(39) µl H2O, 5 µl 10x Buffer, 0.5 µl each primer, 1µl Template, 2µl dNTPs, 1µl Pfu, 0(1) µl DMSO

NO PCR Product

Tuesday, 09/02/2008
No PCR product
 * PCR of LuxQ with taq-Polymerase with different DMSO concentrations (0 %, 5 %, 10 %). As template V. harveyi colonies and supernatant of cooked bacteria was used.

Wednesday, 09/03/2008

 * PCR of LuxP and LuxQ from Vibrio harveyi genome with Phusion Mastermix (without any annealing temp). Template: 0.2 µl V. harveyi culture incubated over night in LB(not 20% NaCl LB) at 37 °C.
 * result: no PCR product
 * PCR of LuxP and LuxQ from Vibrio harveyi genome with Fementas tag Mastermix
 * sample with normal Mg concentration (2 µg) and 6 µg Mg concentration
 * 5min @94°C ||30 sec @ 94°C | 30 sec @ 58°C | 2 min @ 72°C || 5 min @ 72°C | 4°C hold (30 cycles)
 * template is 0.2 ul water washed Vibrio harveyi cells.
 * result: see tomorrow (NO PRODUCT)
 * incubation of Vibrio harveyi from glycerol stock at 28 over night (medium is 20% NaCl LB)

Thursday, 09/04/2008

 * PCR of LuxP and LuxQ from Vibrio hayveyi genome with Tag Mastermix
 * template is 0.3 ul water washed Vibrio harveyi culture from 3rd September sample with DMSO(1 ul 100%DMSO) and without DMSO
 * Result: only byproduct



Friday, 09/05/2008

 * Cloning of LuxQ from day before into DH5α cells with standard protocols
 * Plated over weekend @ room temperature

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