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

From 2008.igem.org

(Difference between revisions)
(→Wednesday, 09/03/2008)
(→Friday, 09/05/2008)
 
(8 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>
 +
h1.firstHeading { display: none; }
 +
 
 +
p {text-align: justify;}
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a:link { color: #00b0e6; text-decoration: none}
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a:visited { color:#00b0e6; text-decoration: none}
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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. */
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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 {
 +
  display: none; /* Hides all side menus. */
 +
}
 +
/*------------------------------------------------------------------------------------- Menu Bar */
 +
div.MenuBar {
 +
  font: arial, helvetica, sans-serif;
 +
  height: 30px;
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  width: 910px;
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  padding: 0;
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  background: black;
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 +
}
 +
div.MenuBar ul {
 +
  font: arial, helvetica, sans-serif;
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  text-align: center;
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  margin: 0.5em auto;
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  padding: 0;
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  background: black;
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div.MenuBar ul li {
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  padding: 0;
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}
 +
 
 +
div.MenuBar ul li a {
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  font: arial, helvetica, sans-serif;
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  display: block;
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  height: 22px; /* Keep height + padding-top + padding-bottom sync with the menu bar height. */
 +
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 +
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 +
  padding-bottom: 4px;
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  padding-right: 1em; /* Sets the right space between top-level items. */
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 +
}
 +
 
 +
/*------------------------------------------------------------------------------ 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.
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                  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. */
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  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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}
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ul.DropDownMenu li a span span {
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  float: left; /* Aligns the text (back) to the left. */
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  font: 12px arial, helvetica, sans-serif; /* Required for IE55. */
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  height: 20px;
 +
  color: #FFFFFF;
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}
 +
/*----------------------------------------------------------------------------------- Side Menus */
 +
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 {
 +
  display: block;
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  width: 11em; /* Side menu width.
 +
                  Use MenuTailor.css to customize. */
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  padding: 1px; /* Sets the side menu "effective border" width. */
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  position: absolute;
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                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. */
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  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;
 +
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}
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div.MenuBar a {
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 +
div.MenuBar ul li:hover a,
 +
div.MenuBar ul li a:hover {
 +
  color: #ea7f16;
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  color: HighlightText;
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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 {
 +
  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;
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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;
 +
}
 +
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">
Line 62: Line 403:
</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">
Line 98: Line 439:
</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]-->
Line 125: Line 467:
== Monday, 09/01/2008 ==
== Monday, 09/01/2008 ==
* 1.selection for Fusion-1 and Fusion-2 cloning
* 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 (Buffer3+BSA at 37)
+
* 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-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
** for Fusion-2: if our insert is there: one band of 6122 bp if no inser: two bands: 4388 + 2213 bp
Line 145: Line 487:
== Wednesday, 09/03/2008 ==
== Wednesday, 09/03/2008 ==
-
* PCR of LuxP and LuxQ from ''Virio 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.
+
* 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
**'''result''': no PCR product
* PCR of LuxP and LuxQ from ''Vibrio harveyi'' genome with Fementas tag Mastermix
* 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
** sample with normal Mg concentration (2 µg) and 6 µg Mg concentration
-
** 5min @94 |30 sec @94||30 sec @58||2 min @72|5 min @72| 4 for ever (30 cycles)
+
** 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.
+
** template is 0.2 ul water washed Vibrio harveyi cells.
**'''result''': see tomorrow ('''NO PRODUCT''')
**'''result''': see tomorrow ('''NO PRODUCT''')
*incubation of ''Vibrio harveyi'' from glycerol stock at 28 over night (medium is '''20% NaCl LB''')
*incubation of ''Vibrio harveyi'' from glycerol stock at 28 over night (medium is '''20% NaCl LB''')
Line 156: Line 498:
== Thursday, 09/04/2008 ==
== Thursday, 09/04/2008 ==
* PCR of LuxP and LuxQ from ''Vibrio hayveyi'' genome with Tag Mastermix
* 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
 +
[[Image:HD 080904-LuxQ PCR.png|center|thumb|400px|LuxQ PCR product]]
[[Image:HD 080904-LuxQ PCR.png|center|thumb|400px|LuxQ PCR product]]
== Friday, 09/05/2008 ==
== Friday, 09/05/2008 ==
-
* Cloning of LuxQ from day before into DH5a cells with standard protocols
+
* Cloning of LuxQ from day before into DH5α cells with standard protocols
-
* Plated over weekend @ RT
+
* Plated over weekend @ room temperature
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/6thweek | Go to 6th week]]
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/6thweek | Go to 6th week]]

Latest revision as of 14:04, 29 October 2008

Back to the overview

Contents

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

  • 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.

No PCR product

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
LuxQ PCR product

Friday, 09/05/2008

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


Go to 6th week

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