Team:Heidelberg/Notebook/Sensing Group/Notebook/10thweek

h1.firstHeading { display: none; }

p {text-align: justify;}

a:link { color: #00b0e6; text-decoration: none} a:visited { color:#00b0e6; text-decoration: none} a:hover { color:#f29400; text-decoration: none} a:active { color:#f29400; text-decoration: none}


 * 1) bodyContent { padding: 10px auto; width: 910px; margin: auto; clear: none; }

table#team_members { text-align: justify; border: 0; } table#team_members h2, table#team_members h3 { clear: both; }

/*---*/ div.MenuBar ul li ul.DropDownMenu { display: none; /* Hides all drop-down menus. */

} /* li:hover works in IE7 and FF2. a:hover works in IE6 and FF2. a:hover breaks li:hover in FF2. div.MenuBar ul li:hover ul.DropDownMenu li ul.SideMenu, div.MenuBar ul li a:hover ul.DropDownMenu li a ul.SideMenu { display: none; /* Hides all side menus. */ } /*- Menu Bar */ div.MenuBar { font: arial, helvetica, sans-serif; height: 30px; width: 910px; /*width: 100%*/ margin: 0; border-top: 0; border-right: 0; border-left: 0; padding: 0; background: black; } div.MenuBar ul { font: arial, helvetica, sans-serif; text-align: center; list-style-type: none; margin: 0.5em auto; border: 0; padding: 0; background: black; } div.MenuBar ul li { font: arial, helvetica, sans-serif; display: block; padding: 0; margin: 0; font-size: 1.3em; float: left; background: black; text-align: center; width: 107px; position: relative; /* Sets the positioning context for each drop-down menu. */ }

div.MenuBar ul li a { font: arial, helvetica, sans-serif; display: block; background: black; height: 22px; /* Keep height + padding-top + padding-bottom sync with the menu bar height. */ color: #ffffff; padding-top: 4px; padding-bottom: 4px; padding-left: 1em; /* Sets the left space between top-level items. */ padding-right: 1em; /* Sets the right space between top-level items. */ text-decoration: none; }

/*-- Drop-Down Menus */ div.MenuBar ul li:hover ul.DropDownMenu, div.MenuBar ul li a:hover ul.DropDownMenu { display: block; width: 10em; /* Drop-down menu width. Use MenuTailor.css to customize. */ height: 1em; padding: 1px; /* Sets the drop-down menu "effective border" width. */ position: absolute; top: 23px; /* Places the drop-down menu under the menu bar. Keep it sync with the menu bar height. */ left: 0; /* Aligns the drop-down menu to its top-level item. */ background-color: black; /* Selected item. */ color: #FFFFFF;

} div.MenuBar ul li:hover ul.DropDownMenu li a, div.MenuBar ul li a:hover ul.DropDownMenu li a { width: 10em; /* Keep it sync with the drop-down menu width. Use MenuTailor.css to customize. */ height: 1em; padding-left: 0; padding-right: 0; background-color: black; /* Selected item. */ color: #FFFFFF; } ul.DropDownMenu li a span { display: block; padding-left: 0.75em; /* Sets the left space of each drop-down menu item. */ padding-right: 0.25em; /* Sets the right space of each drop-down menu item. */ text-align: right; /* Aligns the >> symbol to the right. */ } ul.DropDownMenu li a span span { float: left; /* Aligns the text (back) to the left. */ font: 12px arial, helvetica, sans-serif; /* Required for IE55. */ height: 20px; color: #FFFFFF; } /*--- Side Menus */ div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu, div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu { display: block; width: 11em; /* Side menu width. Use MenuTailor.css to customize. */ padding: 1px; /* Sets the side menu "effective border" width. */ position: absolute; top: -1px; /* Aligns the side menu to its drop-down menu item. Keep it sync with the side menu "effective border" width. */ left: 13em; /* Places the side menu to the right of the drop-down menu. Keep it sync with the drop-down menu width. Use MenuTailor.css to customize. */ } div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a, div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a { width: 11em; /* Keep it sync with the side menu width. Use MenuTailor.css to customize. */ font: 12px arial, helvetica, sans-serif; /* Required for IE55. */ left: 13em; /* Places the side menu to the right of the drop-down menu. Keep it sync with the drop-down menu width. Use MenuTailor.css to customize. */ } div.MenuBar ul li ul.DropDownMenu li ul.SideMenu li a span { padding-left: 1.5em; /* Sets the left space of each side menu item. */ padding-right: 0.5em; /* Sets the right space of each side menu item. */ text-align: left; font: 12px arial, helvetica, sans-serif; /* Required for IE55. */ left: 13em; /* Places the side menu to the right of the drop-down menu. Keep it sync with the drop-down menu width. Use MenuTailor.css to customize. */ } /*- Browser Specific */ float: left; /* Required for IE55 and IE6. Breaks O9. Hidden (* html) from non-IE browsers. */ } cursor: hand; /* Required for IE55. Hidden (* html) from non-IE browsers. */ } ul.DropDownMenu li a:hover { cursor: pointer; /* Required for IE6 and IE7. Hidding it (* html) from non-IE browsers breaks IE7! } text-decoration: none; /* Required for IE55 and IE6. Hidden (* html) from non-IE browsers. */ } border: 0; /* Required for IE55 and IE6. Hidden (* html) from non-IE browsers. */ } /*--- Default Colors */ div.MenuBar { background-color: Menu; border-bottom: 1px solid ButtonShadow; } div.MenuBar a { background-color: Menu; /* Top-level unselected items. */ color: MenuText; } div.MenuBar ul li:hover a, div.MenuBar ul li a:hover { color: #ea7f16; background-color: Highlight; /* Top-level selected item. */ color: HighlightText; } /*...............................................................................................*/ div.MenuBar ul li:hover ul.DropDownMenu, div.MenuBar ul li a:hover ul.DropDownMenu { background-color: ButtonShadow; /* Sets the drop-down menu "effective border" color. */ } div.MenuBar ul li:hover ul.DropDownMenu li a, div.MenuBar ul li a:hover ul.DropDownMenu li a { background-color: Menu; Drop-down menu unselected items. Sets the drop-down menu "effective background" color. */ color: MenuText; } div.MenuBar ul li:hover ul.DropDownMenu li:hover a, div.MenuBar ul li a:hover ul.DropDownMenu li a:hover { background-color: Highlight; /* Drop-down menu selected item. */ color: HighlightText; } /*...............................................................................................*/ div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu, div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu { background-color: ButtonShadow; /* Sets the side menu "effective border" color. */ } div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a, div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a { background-color: Menu; /* Side menu unselected items. Sets the side menu "effective background" color. */ color: MenuText; } div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a:hover, div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a:hover { background-color: Highlight; /* Side menu selected item. */ color: HighlightText; } /*---*/ /*Script-Free 3-Level Menu 1.2 Tailor www.CesarDaniel.info /*-- General */ body { background: white; color: black; margin: 0; border: 0; padding: 0; }
 * html div.MenuBar ul li a {
 * html ul.DropDownMenu li a:hover {
 * html div.MenuBar a:hover {
 * html div.MenuBar ul li table,
 * html div.MenuBar ul li table td {

div.MenuBar { font: 13px arial, helvetica, sans-serif; } div.MenuBar ul { font: 13px arial, helvetica, sans-serif; /* Required for IE55. */ } /*--- Colors */ div.MenuBar { background-color: black; /* Selected item. */ color: #FFFFFF; border-bottom: 1px solid ButtonShadow; } div.MenuBar a, div.MenuBar ul li:hover ul.DropDownMenu li a, div.MenuBar ul li a:hover ul.DropDownMenu li a, div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a, div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a { background-color: black; /* Selected item. */ color: #FFFFFF; } div.MenuBar ul li:hover a, div.MenuBar ul li a:hover, div.MenuBar ul li:hover ul.DropDownMenu li:hover a, div.MenuBar ul li a:hover ul.DropDownMenu li a:hover, div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a:hover, div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a:hover { background-color: #00b0e6; /* Selected item. */ color: #FFFFFF; } div.MenuBar ul li:hover ul.DropDownMenu, div.MenuBar ul li a:hover ul.DropDownMenu, div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu, div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu { background-color: ButtonShadow; /* Sets the drop-down and side menus "effective border" color. */ } /*--- Widths */ /*

/* Menu Bar 1 Drop-Down Menu #2 div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4, div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4, div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM4 li a, div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM4 li a { width: 11em; /* Drop-down menu width. */ } div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5, div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5, div.MenuBar#navi ul li:hover ul.DropDownMenu#MB1-DDM5 li a, div.MenuBar#navi ul li a:hover ul.DropDownMenu#MB1-DDM5 li a { width: 12em; /* Drop-down menu width. */ }

/*...............................................................................................*/ /* Menu Bar 1 Drop-Down Menu #2 Side Menu #1 div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1, div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 { left: 15.5em !important; /* Places the side menu to the right of the drop-down menu. Keep it sync with the drop-down menu width. */ } div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1, div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1, div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1 li a, div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 li a { width: 10em; /* Side menu width. */ } /*...............................................................................................*/ /* Menu Bar 1 Drop-Down Menu #2 Side Menu #2 div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2, div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 { left: 15.5em !important; /* Places the side menu to the right of the drop-down menu. Keep it sync with the drop-down menu width. */ } div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2, div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2, div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2 li a, div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 li a { width: 10em; /* Side menu width. */ } /*...............................................................................................*/ /* Menu Bar 1 Drop-Down Menu #2 Side Menu #3 div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3, div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 { left: 15.5em !important; /* Places the side menu to the right of the drop-down menu. Keep it sync with the drop-down menu width. */ } div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3, div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3, div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3 li a, div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 li a { width: 10em; /* Side menu width. */ } /*...............................................................................................*/

   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>

<div id="header_bottom"><img src="http://2008.igem.org/wiki/images/e/ef/Navi_bg.gif" alt="" / >

Back to the overview

Monday, 10/06/2008
--> No PCR product
 * Mutagenesis PCR of LuxS,P with Phusion and the following programme (20 cycles)
 * PCR programme: 30 sec @ 98°C || 10 sec @ 98 °C | 30 sec @ 55 °C | 30 sec @ 72 °C || 5 min @ 72 °C | 4°C

Tuesday, 10/07/2008
--> only LuxS2 positive --> Gelextraction
 * Repetition of Mutagenesis PCR with Phusion and same programme, but 30 cycles


 * New PCR with Turbo Pfu and iProof (18 cycles)
 * 5(10) µl buffer
 * 1 µl forward Primer (10 µM stock)
 * 1 µl reverse Primer
 * 1 µl dNTPs
 * 2 µl Template
 * 39(34) µl water
 * 1 µl Polymerase
 * 30 sec @ 95 °C ||30 sec @ 95 °C | 60 sec @ 55 °C | 30 sec @ 68 °C || 8 min @ 68 °C | 4°C hold

Wednesday, 10/08/2008

 * Miniprep of LuxS
 * New PCR of LuxS and LuxP with Cloned Pfu and Phusion Polymerase
 * Cloned Pfu:
 * 30s @ 95°C || 30s @ 95°C | 1min @ 55°C | 1min @ 68°C || 8min @ 72°C | 4min (20 cycles)


 * Phusion:
 * 30s @ 98°C || 10s @ 98°C | 1min @ 72°C || 5min @ 72°C | 4°C (25 cycles)

-->NO PCR Product


 * New PCR with Taq:
 * 2min @ 95°C || 30s @ 95°C | 30s @ 55°C | 1min @ 72°C || 10 min @ 72°C | 4°C (20 cycles)

-->NO PCR Product

Check Templates tomorrow, by digestion and undigested

Thursday, 10/09/2008

 * Digestion of LuxS with (BamHI/NcoI and BamHI/EcoRI) and LuxP (NcoI) both negative
 * O/N culture of LuxS
 * Cloning of Tar-YFP from pDK58
 * Digestion of pDK58, F1-LuxP-pDK48, F2-LuxP-pDK58 with KpnI/NotI
 * Gelextraction eluted in 30 µl H2O
 * Ligation of 5µl Insert with 5µl Vector 30 min @ 30°C
 * Transformation into DH5a

Friday, 10/10/2008



 * LuxS digestion with BamHI/EcoRI (NEBuffer EcoRI + BSA) to check for insert
 * LuxS mutagenesis PCR for EcoRI did not work
 * Prefix/Suffix PCR of GeneArt Construct
 * PCR of LuxS1, LuxS2, LuxS3 with Phusion. Annealing Temperature Gradient 50-60°C

Go to 11th week