Team:Heidelberg/Notebook/Sensing Group/Notebook/1stweek

From 2008.igem.org

(Difference between revisions)
(→Cloning of LuxS)
(→Friday, 08/08/2008)
 
(14 intermediate revisions not shown)
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<html>
<html>
-
<link rel='stylesheet' href='http://igem-heidelberg.de/fileadmin/Wiki/Heidelberg2.css' type='text/css' />
+
 
-
<link rel="stylesheet" href="http://igem-heidelberg.de/fileadmin/Wiki/Menu.css" type="text/css"/>
+
 
-
<link rel="stylesheet"  href="http://igem-heidelberg.de/fileadmin/Wiki/MenuHD.css" type="text/css"/>
+
<style>
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h1.firstHeading { display: none; }
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p {text-align: justify;}
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a:visited { color:#00b0e6; text-decoration: none}
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a:hover { color:#f29400; text-decoration: none}
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a:active { color:#f29400; text-decoration: none}
 +
 
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#bodyContent { padding: 10px auto; width: 910px; margin: auto; clear: none; }
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table#team_members { text-align: justify; border: 0; }
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table#team_members h2, table#team_members h3 { clear: both; }
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div.MenuBar ul li ul.DropDownMenu {
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/*
 +
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 {
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  display: none; /* Hides all side menus. */
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}
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/*------------------------------------------------------------------------------------- Menu Bar */
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div.MenuBar {
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  font: arial, helvetica, sans-serif;
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div.MenuBar ul li a {
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}
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 +
/*------------------------------------------------------------------------------ Drop-Down Menus */
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div.MenuBar ul li:hover ul.DropDownMenu,
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div.MenuBar ul li a:hover ul.DropDownMenu {
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                  Use MenuTailor.css to customize. */
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  padding: 1px; /* Sets the drop-down menu "effective border" width. */
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  position: absolute;
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  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;
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 +
}
 +
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. */
 +
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  padding-right: 0;
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  background-color: black; /* Selected item. */
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  color: #FFFFFF;
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}
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ul.DropDownMenu li a span {
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  display: block;
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  text-align: right; /* Aligns the >> symbol to the right. */
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}
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ul.DropDownMenu li a span span {
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  color: #FFFFFF;
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}
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/*----------------------------------------------------------------------------------- Side Menus */
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div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,
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div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {
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                Keep it sync with the side menu "effective border" width. */
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                            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 {
 +
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  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 */
 +
* html div.MenuBar ul li a {
 +
  float: left; /* Required for IE55 and IE6.
 +
                            Breaks O9.
 +
                            Hidden (* html) from non-IE browsers. */
 +
}
 +
* html ul.DropDownMenu li a:hover {
 +
  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!
 +
}
 +
* html div.MenuBar a:hover {
 +
  text-decoration: none; /* Required for IE55 and IE6.
 +
                            Hidden (* html) from non-IE browsers. */
 +
}
 +
* html div.MenuBar ul li table,
 +
* html div.MenuBar ul li table td {
 +
  border: 0; /* Required for IE55 and IE6.
 +
                Hidden (* html) from non-IE browsers. */
 +
}
 +
/*------------------------------------------------------------------------------- Default Colors */
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div.MenuBar ul li:hover a,
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div.MenuBar ul li a:hover {
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}
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/*...............................................................................................*/
 +
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 {
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 +
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 +
}
 +
/*...............................................................................................*/
 +
div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu,
 +
div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu {
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}
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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;
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  margin: 0;
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  border: 0;
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  padding: 0;
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}
 +
 
 +
 
 +
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;
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}
 +
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;
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}
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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,
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div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a:hover,
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div.MenuBar ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu li a:hover {
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  background-color: #00b0e6; /* Selected item. */
 +
  color: #FFFFFF;
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}
 +
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
 +
*/
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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. */
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}
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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. */
 +
}
 +
/*...............................................................................................*/
 +
 
 +
</style>
 +
 
<body>
<body>
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</li>
</li>
<li>
<li>
-
<a href="https://2008.igem.org/Team:Heidelberg/Chemotaxis" style="color: white">Modeling<!--[if gt IE 6]><!--></a><!--<![endif]-->
+
<a href="https://2008.igem.org/Team:Heidelberg/Modeling" style="color: white">Modeling<!--[if gt IE 6]><!--></a><!--<![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<ul class="DropDownMenu" id="MB1-DDM3">
<ul class="DropDownMenu" id="MB1-DDM3">
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</li>
</li>
<li>
<li>
-
<a href="https://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook" style="color: white">Notebook<!--[if gt IE 6]><!--></a><!--<![endif]-->
+
<a href="https://2008.igem.org/Team:Heidelberg/Notebook/Overview" style="color: white">Notebook<!--[if gt IE 6]><!--></a><!--<![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<ul class="DropDownMenu" id="MB1-DDM5">
<ul class="DropDownMenu" id="MB1-DDM5">
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</li>
</li>
<li style="width: 160px">
<li style="width: 160px">
-
<a href="https://2008.igem.org/Team:Heidelberg/Project/General_information" style="color: white">Human Practice<!--[if gt IE 6]><!--></a><!--<![endif]-->
+
<a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview" style="color: white">Human Practice<!--[if gt IE 6]><!--></a><!--<![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<!--[if lt IE 7]><table border="0" cellpadding="0" cellspacing="0"><tr><td><![endif]-->
<ul class="DropDownMenu" id="MB1-DDM4">
<ul class="DropDownMenu" id="MB1-DDM4">
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                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"><span><span>Surveys</span></span></a></li>
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"><span><span>Surveys</span></span></a></li>
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"><span><span>Open Day</span></span></a></li>
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"><span><span>Open Day</span></span></a></li>
-
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"><span><span>Nobel Prize</span></span></a></li>
+
                               <li><a href="https://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"><span><span>Nobel Prize</span></span></a></li>
 +
</ul>
</ul>
<!--[if lte IE 6]></td></tr></table></a><![endif]-->
<!--[if lte IE 6]></td></tr></table></a><![endif]-->
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=== Preparations ===
=== Preparations ===
Preparations were started at the end of the week before
Preparations were started at the end of the week before
-
* Transformation of pDK48 and pTrc99alpha plamids in ''E. coli'' strain DH5alpha (0.5 µl plasmid-DNA + 50 µl competent cells)
+
* Transformation of pDK48 and pTrc99alpha plamids in ''E. coli'' strain DH5α (0.5 µl plasmid-DNA + 50 µl competent cells)
* Glycerol-stock of ''Vibrio harveyi'' BB120, BB886, mm30, BB178, BB125
* Glycerol-stock of ''Vibrio harveyi'' BB120, BB886, mm30, BB178, BB125
-
* pick pTrc99a and pDK48 colonies and then incubated in 3 ml LB at 37 overnight
+
* pick pTrc99α and pDK48 colonies and then incubated in 3 ml LB at 37 overnight
* ''V. harveyi'' cultured on LB plate
* ''V. harveyi'' cultured on LB plate
-
* MiniPrep of pTrc99a and pDK48 from DH5alpha
+
* MiniPrep of pTrc99α and pDK48 from DH5α
== Tuesday, 08/05/2008 ==
== Tuesday, 08/05/2008 ==
=== Cloning of LuxP ===
=== Cloning of LuxP ===
[[Image:HD_080805-LuxP_PCR.png|thumb|150px|LuxP colony PCR]]
[[Image:HD_080805-LuxP_PCR.png|thumb|150px|LuxP colony PCR]]
-
<div style="float: right; clear:none">[[Image:HD 080805-LuxQ S PCR.png|right|thumb|200px|LuxQ, LuxS PCR]]</div>
+
<div style="float: right; clear:none;">[[Image:HD 080805-LuxQ S PCR.png|right|thumb|200px|LuxQ, LuxS PCR]]</div>
-
* PCR of LuxP from ''V. harveyi'' colony with the primers LuxPc and LuxPd @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles) with Tag polymerase Mastermix
+
 
 +
* PCR of LuxP from ''V. harveyi'' colony with the primers LuxPc and LuxPd with Taq polymerase Mastermix. 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
* Gel Purification of LuxP eluted in 30 µl H<sub>2</sub>O
* Gel Purification of LuxP eluted in 30 µl H<sub>2</sub>O
* Digestion of LuxP with SalI and NotI of pDK48 and LuxP-Gel-Purification product: 2 µl NEB-Buffer 3 + 0.5 µl SalI & NotI + 10 µl DNA + 6 µl H<sub>2</sub>O
* Digestion of LuxP with SalI and NotI of pDK48 and LuxP-Gel-Purification product: 2 µl NEB-Buffer 3 + 0.5 µl SalI & NotI + 10 µl DNA + 6 µl H<sub>2</sub>O
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=== Cloning of LuxS ===
=== Cloning of LuxS ===
-
* PCR of ''V. harveyi'' genome with primers LuxSa and LuxSb and PCR purification 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles) with Tag polymerase Mastermix  
+
* PCR of ''V. harveyi'' genome with primers LuxSa and LuxSb and PCR purification 5min @ 95 °C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles) with Tag polymerase Mastermix  
-
* digestion of LuxS and pTrc99a with BamHI/NcoI  
+
* digestion of LuxS and pTrc99α with BamHI/NcoI (NEBuffer 3 + BSA)
* Ligation with vector:insert ratio 2µl:6µl and 5µl:5µl
* Ligation with vector:insert ratio 2µl:6µl and 5µl:5µl
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== Wednesday, 08/06/2008 ==
== Wednesday, 08/06/2008 ==
-
* 5 colonies of LuxQ,S,P transformation (1:1 ligation) picket and used for colony-PCR
+
* 5 colonies of LuxQ,S,P transformation (1:1 ligation) picked and analyes with colony-PCR
-
* Colony-PCR to check for insert. 5min @ 94°C || 30s @ 94°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C  | 4°C hold (30 cycles)
+
* Colony-PCR to check for insert. 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C  | 4°C hold (30 cycles)
[[Image:HD 080806-LuxQ P S ColonyPCR.png|center|thumb|500px|Colony PCR of LuxQ, LuxS and LuxP]]
[[Image:HD 080806-LuxQ P S ColonyPCR.png|center|thumb|500px|Colony PCR of LuxQ, LuxS and LuxP]]
* preparation of O/N cultures for Miniprep
* preparation of O/N cultures for Miniprep
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* PCR from Minipreps to confirm inserts. Same programme as yesterday
* PCR from Minipreps to confirm inserts. Same programme as yesterday
[[Image:HD 080807-LuxQ P S miniPCR.png|center|thumb|500px|PCR to confirm insert from Minipreps. LuxQ no. 3 seems to be posivite. For LuxS and LuxP all samples are positive.]]
[[Image:HD 080807-LuxQ P S miniPCR.png|center|thumb|500px|PCR to confirm insert from Minipreps. LuxQ no. 3 seems to be posivite. For LuxS and LuxP all samples are positive.]]
-
* 10 new colonies of LuxQ-plate picked and checked via colony-PCR
+
* 10 new colonies of LuxQ-plate were picked and checked via colony-PCR
[[Image:HD_080807-LuxQ_ColonyPCR.png|center|thumb|500px|LuxQ colony PCR of 10 new colonies.]]
[[Image:HD_080807-LuxQ_ColonyPCR.png|center|thumb|500px|LuxQ colony PCR of 10 new colonies.]]
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* ordered Sequencing of LuxS,P,Q positive clones at GATC
+
* Sequencing of LuxS,P,Q positive clones at GATC
  Sequencing Results
  Sequencing Results
  LuxS: correct sequence
  LuxS: correct sequence
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== Friday, 08/08/2008 ==
== Friday, 08/08/2008 ==
* Miniprep of overnight-cultures of the picked colonies Q#6-15
* Miniprep of overnight-cultures of the picked colonies Q#6-15
-
* In silico cloning of LuxQ, S in pTrc99a and LuxP in pDK48
+
* In silico cloning of LuxQ, S in pTrc99α and LuxP in pDK48
* Test-Digestion of LuxQ with XbaI --> positive result for Q3, 7, 9, 11, 14
* Test-Digestion of LuxQ with XbaI --> positive result for Q3, 7, 9, 11, 14
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/2ndweek | Go to 2nd week]]
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/2ndweek | Go to 2nd week]]

Latest revision as of 14:01, 29 October 2008

Back to the overview

Contents

Monday, 08/04/2008

Preparations

Preparations were started at the end of the week before

  • Transformation of pDK48 and pTrc99alpha plamids in E. coli strain DH5α (0.5 µl plasmid-DNA + 50 µl competent cells)
  • Glycerol-stock of Vibrio harveyi BB120, BB886, mm30, BB178, BB125
  • pick pTrc99α and pDK48 colonies and then incubated in 3 ml LB at 37 overnight
  • V. harveyi cultured on LB plate
  • MiniPrep of pTrc99α and pDK48 from DH5α

Tuesday, 08/05/2008

Cloning of LuxP

LuxP colony PCR
LuxQ, LuxS PCR
  • PCR of LuxP from V. harveyi colony with the primers LuxPc and LuxPd with Taq polymerase Mastermix. 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
  • Gel Purification of LuxP eluted in 30 µl H2O
  • Digestion of LuxP with SalI and NotI of pDK48 and LuxP-Gel-Purification product: 2 µl NEB-Buffer 3 + 0.5 µl SalI & NotI + 10 µl DNA + 6 µl H2O
  • Ligation with vector:insert ratio 2µl:6µl and 5µl:5µl

Cloning of LuxS

  • PCR of V. harveyi genome with primers LuxSa and LuxSb and PCR purification 5min @ 95 °C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles) with Tag polymerase Mastermix
  • digestion of LuxS and pTrc99α with BamHI/NcoI (NEBuffer 3 + BSA)
  • Ligation with vector:insert ratio 2µl:6µl and 5µl:5µl


Wednesday, 08/06/2008

  • 5 colonies of LuxQ,S,P transformation (1:1 ligation) picked and analyes with colony-PCR
  • Colony-PCR to check for insert. 5min @ 95°C || 30s @ 95°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
Colony PCR of LuxQ, LuxS and LuxP
  • preparation of O/N cultures for Miniprep

Thursday, 08/07/2008

  • Miniprep of samples Q#1-5, S#1-5, P#1-5
  • PCR from Minipreps to confirm inserts. Same programme as yesterday
PCR to confirm insert from Minipreps. LuxQ no. 3 seems to be posivite. For LuxS and LuxP all samples are positive.
  • 10 new colonies of LuxQ-plate were picked and checked via colony-PCR
LuxQ colony PCR of 10 new colonies.
  • Sequencing of LuxS,P,Q positive clones at GATC 
Sequencing Results
LuxS: correct sequence
LuxP: all clones contain mutations, cloning has to be repeated
LuxQ: no insert in the picked colonies

Friday, 08/08/2008

  • Miniprep of overnight-cultures of the picked colonies Q#6-15
  • In silico cloning of LuxQ, S in pTrc99α and LuxP in pDK48
  • Test-Digestion of LuxQ with XbaI --> positive result for Q3, 7, 9, 11, 14


Go to 2nd week

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