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

From 2008.igem.org

(Difference between revisions)
(→Tuesday, 08/12/2008)
(→Friday, 08/15/2008)
 
(10 intermediate revisions not shown)
Line 1: Line 1:
<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>
 +
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}
 +
 
 +
#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;
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  /*width: 100%*/
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  border-right: 0;
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  border-left: 0;
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  padding: 0;
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  background: black;
 +
 
 +
}
 +
div.MenuBar ul {
 +
  font: arial, helvetica, sans-serif;
 +
  text-align: center;
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  margin: 0.5em auto;
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  border: 0;
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  padding: 0;
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  background: black;
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}
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div.MenuBar ul li {
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  font: arial, helvetica, sans-serif;
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  padding: 0;
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  margin: 0;
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  float: left;
 +
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  width: 107px;
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  position: relative; /* Sets the positioning context for each drop-down menu. */
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}
 +
 
 +
div.MenuBar ul li a {
 +
  font: arial, helvetica, sans-serif;
 +
  display: block;
 +
  background: black;
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  height: 22px; /* Keep height + padding-top + padding-bottom sync with the menu bar height. */
 +
  color: #ffffff;
 +
  padding-top: 4px;
 +
  padding-bottom: 4px;
 +
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  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. */
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  text-align: right; /* Aligns the >> symbol to the right. */
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}
 +
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 */
 +
* 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 */
 +
div.MenuBar {
 +
  background-color: Menu;
 +
  border-bottom: 1px solid ButtonShadow;
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}
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div.MenuBar a {
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  background-color: Menu; /* Top-level unselected items. */
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  color: MenuText;
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}
 +
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;
 +
}
 +
 
 +
 
 +
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. */
 +
}
 +
/*...............................................................................................*/
 +
 
 +
</style>
 +
 
<body>
<body>
Line 52: Line 393:
</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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== Tuesday, 08/12/2008 ==
== Tuesday, 08/12/2008 ==
* Miniprep of correctly sequenced LuxS and Glycerol-stock
* Miniprep of correctly sequenced LuxS and Glycerol-stock
-
* LuxQ no. 3 checked via digestion with NcoI/BamHI --> negative result
+
* 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
* PCR for LuxQ, LuxQ-1, LuxQ-2, Tar-1, Tar-2 with Taq Mastermix
-
** 5min @ 95°C || 30s @ 94°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)
+
** 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
* preparation of O/N culture for LuxQ no. 3
== Wednesday, 08/13/2008 ==
== Wednesday, 08/13/2008 ==
-
* PCR-purification of the 5 PCR-products eluted in 50 µl H<sub>2</sub>O
+
* PCR-purification of the all PCR-products eluted in 50 µl H<sub>2</sub>O
-
* Miniprep of LuxQ no. 3 O/N culture and digestion with xbaI
+
* Miniprep of LuxQ no. 3 O/N culture and digestion with xbaI (NEBuffer 2 + BSA)
-
* Fusion-PCR for Fusion-1 and repetetion of PCR for LuxQ-2 and LuxQ
+
* Fusion-PCR for Fusion-1 and repetition of PCR for LuxQ-2 and LuxQ
-
* digestion of pDK48 and Fusion-1 with NcoI/NdeI (0.5 µl) and AgeI (1 µl) in NEBuffer 4
+
* digestion of pDK48 plasmid and Fusion-1 frament with NcoI/NdeI (0.5 µl) and AgeI (1 µl) in NEBuffer 4
-
<div style="float: left;>[[Image:HD 080813-LuxQ PCR-pDK48 mini LuxQ3 xbaI.png|thumb|500px|Fusion-PCR for Fusion-1, LuxQ PCR, LuxQ-2 PCR, pDK48 miniprep and xbaI digestion of LuxQ no. 3]]</div>
+
[[Image:HD 080813-LuxQ PCR-pDK48 mini LuxQ3 xbaI.png|left|thumb|500px|Fusion-PCR for Fusion-1, LuxQ PCR, LuxQ-2 PCR, pDK48 miniprep and xbaI digestion of LuxQ no. 3]] [[Image:HD 080813-F1 pDK48 Digestion.png|left|thumb|200px|Digestion of Fusion-1 and pDK48 with NcoI/NdeI/AgeI]]
-
<div style="float: left;">[[Image:HD 080813-F1 pDK48 Digestion.png|thumb|200px|Digestion of Fusion-1 and pDK48 with NcoI/NdeI/AgeI]]</div>
+
<br style="clear: both;"/>
<br style="clear: both;"/>
Line 145: Line 486:
== Thursday, 08/14/2008 ==
== Thursday, 08/14/2008 ==
[[Image:HD 080814-LuxQ PCR.png|thumb|right|PCR for LuxQ and LuxQ-2]]
[[Image:HD 080814-LuxQ PCR.png|thumb|right|PCR for LuxQ and LuxQ-2]]
-
* Gel from LuxQ, LuxQ2
+
* Gel for LuxQ, LuxQ2
* sequentiell digestion of Fusion-1 with NcoI and NdeI (NEBuffer 3)
* sequentiell digestion of Fusion-1 with NcoI and NdeI (NEBuffer 3)
* Double digestion of pDK48 with NcoI/NdeI (NEBuffer 4)
* Double digestion of pDK48 with NcoI/NdeI (NEBuffer 4)
Line 153: Line 494:
== Friday, 08/15/2008 ==
== Friday, 08/15/2008 ==
* digestion of Fusion-1 with AgeI in NEBuffer 1
* digestion of Fusion-1 with AgeI in NEBuffer 1
-
* Gelextraction of sequentially digested Fusion-1 and pDK48
+
* gel extraction of sequentially digested Fusion-1 and pDK48 products
-
* new digestion of Fusion-1 with NcoI/NdeI and Gelextraction, because previous digestion did not yield expected bands (there should be two bands at 516bp and 392bp)
+
* 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)
[[Image:HD 080815-F1 digestion pDK48.png|left|thumb|250px|Gelextraction of digested Fusion-1 and purified PCR product as well as pDK48 digested. Expected bands at 516bp and 392bp are not visible.]]
[[Image:HD 080815-F1 digestion pDK48.png|left|thumb|250px|Gelextraction of digested Fusion-1 and purified PCR product as well as pDK48 digested. Expected bands at 516bp and 392bp are not visible.]]
[[Image:HD 080815-F1 digestion NcoI NdeI.png|left|thumb|200px|Fusion-1 digested with NcoI/NdeI. Expected bands of 1025bp and 908bp had to be cut out together. Wanted band is the longer one.]]
[[Image:HD 080815-F1 digestion NcoI NdeI.png|left|thumb|200px|Fusion-1 digested with NcoI/NdeI. Expected bands of 1025bp and 908bp had to be cut out together. Wanted band is the longer one.]]
Line 160: Line 501:
<br style="clear: both;"/>
<br style="clear: both;"/>
-
* Ligation of 5 µl Fusion-1, digested with NcoI/NdeI and 5 µl pDK48
+
* Ligation of 5 µl Fusion-1 with 5 µl pDK48 (both digested with NcoI/NdeI )
-
* Transformation into DH5a (5 µl Ligation & 15 µl Ligation)
+
* Transformation into DH5α (5 µl Ligation products)
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/3rdweek | Go to 3rd week]]
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/3rdweek | Go to 3rd week]]

Latest revision as of 14:02, 29 October 2008

Back to the overview

Contents

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
Fusion-PCR for Fusion-1, LuxQ PCR, LuxQ-2 PCR, pDK48 miniprep and xbaI digestion of LuxQ no. 3
Digestion of Fusion-1 and pDK48 with NcoI/NdeI/AgeI


Thursday, 08/14/2008

PCR for LuxQ and LuxQ-2
  • Gel for LuxQ, LuxQ2
  • sequentiell digestion of Fusion-1 with NcoI and NdeI (NEBuffer 3)
  • Double digestion of pDK48 with NcoI/NdeI (NEBuffer 4)


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)
Gelextraction of digested Fusion-1 and purified PCR product as well as pDK48 digested. Expected bands at 516bp and 392bp are not visible.
Fusion-1 digested with NcoI/NdeI. Expected bands of 1025bp and 908bp had to be cut out together. Wanted band is the longer one.


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


Go to 3rd week

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