Team:Heidelberg/Notebook/Sensing Group/Notebook/6thweek

From 2008.igem.org

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(→Fusion chimeras)
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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;}
 +
 
 +
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;
 +
  /*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 */
 +
* 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;
 +
}
 +
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;
 +
}
 +
 
 +
 
 +
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>
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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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== Monday, 09/08/2008 ==
== Monday, 09/08/2008 ==
-
* Liquid Culture of 5 colonies from LuxQ Transformation and 1 from F1 transformation
+
* Culture of 5 colonies from LuxQ Transformation and 1 from F1 transformation
-
* Miniprep of Liquid Culture from previous Transformation (done by Chenchen). Incubated for two days @ RT and about 6 h @ 37 °C
+
* Miniprep of Culture from previous Transformation (done by Chenchen). Incubated for two days @ RT and about then 6 h @ 37 °C
-
* Digestion of the Miniprep products with xbaI (Buffer2 + BSA, 1 ul enzyme @ 37 °C about 40min)
+
* Digestion of the Miniprep products with XbaI (NEBuffer 2 + BSA, 1 ul enzyme @ 37 °C about 40min)
[[Image:HD 080908-LuxQ Colonies-Digestion xbaI.png|center|thumb|500px|Digestion of LuxQ with xbaI yielded expected bands (4736 + 1204 bp) for all samples, except for no. 4]]
[[Image:HD 080908-LuxQ Colonies-Digestion xbaI.png|center|thumb|500px|Digestion of LuxQ with xbaI yielded expected bands (4736 + 1204 bp) for all samples, except for no. 4]]
-
  Sequencing @ GATC yielded correct sequence for LuxQ no. 1
+
  Sequencing @ GATC verified correct sequence for LuxQ no. 1
* PCR for LuxQ and LuxP with Phusion (25 µl Mastermix, 0.5 µl each Primer, 0(1) µl DMSO, 24(23) µl H<sub>2</sub>0, 0.5 µl Template)
* PCR for LuxQ and LuxP with Phusion (25 µl Mastermix, 0.5 µl each Primer, 0(1) µl DMSO, 24(23) µl H<sub>2</sub>0, 0.5 µl Template)
* PCR Programme: 5 min @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C | 30 s @ 72 °C || 5 min @ 72 °C | 4 °C  
* PCR Programme: 5 min @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C | 30 s @ 72 °C || 5 min @ 72 °C | 4 °C  
Line 154: Line 496:
=== LuxP cloning ===
=== LuxP cloning ===
-
* Miniprep of O/N LuxP cultures and digestion with NcoI (2h @ 37 °C)  
+
* Miniprep of O/N LuxP cultures and digestion with NcoI (NEBuffer 2, 2h @ 37 °C)  
[[Image:HD 080910-LuxP Digestion NcoI.png|center|thumb|400px|LuxP digestion with NcoI. All colonies are positive]]
[[Image:HD 080910-LuxP Digestion NcoI.png|center|thumb|400px|LuxP digestion with NcoI. All colonies are positive]]
 +
Sequencing @ GATC: LuxP1 correct
=== Fusion chimeras ===
=== Fusion chimeras ===
* PCR for LuxQ 1+2, Tar 1+2 and Gel Extraction to get rid of first primers
* PCR for LuxQ 1+2, Tar 1+2 and Gel Extraction to get rid of first primers
* Fusion-PCR of LuxQ1+Tar1 und LuxQ2+Tar2 with Phusion and 55 °C annealing --> No product
* Fusion-PCR of LuxQ1+Tar1 und LuxQ2+Tar2 with Phusion and 55 °C annealing --> No product
-
* PCR of LuxQ fragment 1b, LuxQ fragment 1c, LuxQ fragment 2b, LuxQ fragment 2c, Tar fragment 1b, Tar fragment 1c, Tar fragment 2b, Tar fragment 2c with Phusion and 55 °C annealing. Products were purified via Gelextraction to get rid of first primers
+
* Fusion PCR of LuxQ fragment 1b, LuxQ fragment 1c, LuxQ fragment 2b, LuxQ fragment 2c, Tar fragment 1b, Tar fragment 1c, Tar fragment 2b, Tar fragment 2c with Phusion and 55 °C annealing. Products were purified via Gelextraction to get rid of first primers
* Two Fusion PCR (30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)) with PCR purification.
* Two Fusion PCR (30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)) with PCR purification.
* Gel Extraction of LuxQ 1+2, Tar 1+2
* Gel Extraction of LuxQ 1+2, Tar 1+2
-
[[Image:HD 080910-F1 2 Phusion2.png|left|thumb|200px|First Fusion PCR]][[Image:HD 080910-Fusion PCR 2.png|left|thumb|another fusion PCR]]
+
[[Image:HD 080910-F1 2 Phusion2.png|left|thumb|200px|First Fusion PCR]][[Image:HD 080910-Fusion PCR 2.png|left|thumb|400px|another fusion PCR]]
 +
 
 +
<br style="clear: both;" />
== Thursday, 09/11/2008 ==
== Thursday, 09/11/2008 ==
 +
 +
=== Fusion chimeras ===
 +
* PCR Purification of previous Fusion PCR
 +
* Digestion with NcoI/NdeI (NEBuffer4) and NcoI/KpnI (NEBuffer1 + BSA). 1 h @ 37 °C
 +
* Gel extraction, eluted in 30 µl water
 +
* Ligation (Insert:Vector 5 µl:5 µl) and transformation into DH5alpha competent cells
== Friday, 09/12/2008 ==
== Friday, 09/12/2008 ==
 +
nothing to report
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/7thweek | Go to 7th week]]
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/7thweek | Go to 7th week]]

Latest revision as of 18:24, 28 October 2008

Back to the overview

Contents

Monday, 09/08/2008

  • Culture of 5 colonies from LuxQ Transformation and 1 from F1 transformation
  • Miniprep of Culture from previous Transformation (done by Chenchen). Incubated for two days @ RT and about then 6 h @ 37 °C
  • Digestion of the Miniprep products with XbaI (NEBuffer 2 + BSA, 1 ul enzyme @ 37 °C about 40min)
Digestion of LuxQ with xbaI yielded expected bands (4736 + 1204 bp) for all samples, except for no. 4
Sequencing @ GATC verified correct sequence for LuxQ no. 1
  • PCR for LuxQ and LuxP with Phusion (25 µl Mastermix, 0.5 µl each Primer, 0(1) µl DMSO, 24(23) µl H20, 0.5 µl Template)
  • PCR Programme: 5 min @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C | 30 s @ 72 °C || 5 min @ 72 °C | 4 °C
PCR for LuxQ and LuxP. LuxQ PCR did not work, but first PCR for LuxP was positive.

Tuesdsay, 09/09/2008

  • Miniprep of LuxQ colonies no. 1,2,3,5,6
HD 080909-PCR Miniprep-LuxQ 2.png
HD 080909-PCR Miniprep-LuxQ 1.png


  • PCR for LuxQ fragment 1 and 2, Tar fragment 1 and 2 for Phusion receptor with Phusion-Polymerase
  • PCR Programme: 30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)
PCR of LuxQ and Tar constructs for fusion receptor. LuxQ is negative
PCR of LuxQ constructs for fusion


  • Fusion-PCR for Fusion-1 and Fusion-2 constructs
Fusion-PCR
  • O/N Culture of 10 LuxP in pDK48 colonies

Wednesday, 09/10/2008

LuxP cloning

  • Miniprep of O/N LuxP cultures and digestion with NcoI (NEBuffer 2, 2h @ 37 °C)
LuxP digestion with NcoI. All colonies are positive
Sequencing @ GATC: LuxP1 correct

Fusion chimeras

  • PCR for LuxQ 1+2, Tar 1+2 and Gel Extraction to get rid of first primers
  • Fusion-PCR of LuxQ1+Tar1 und LuxQ2+Tar2 with Phusion and 55 °C annealing --> No product
  • Fusion PCR of LuxQ fragment 1b, LuxQ fragment 1c, LuxQ fragment 2b, LuxQ fragment 2c, Tar fragment 1b, Tar fragment 1c, Tar fragment 2b, Tar fragment 2c with Phusion and 55 °C annealing. Products were purified via Gelextraction to get rid of first primers
  • Two Fusion PCR (30 s @ 98 °C || 10 s @ 98 °C | 30 s @ 55 °C |30 s @ 72 °C || 5 min @ 72 °C | 4 °C (30 cycles)) with PCR purification.
  • Gel Extraction of LuxQ 1+2, Tar 1+2
First Fusion PCR
another fusion PCR


Thursday, 09/11/2008

Fusion chimeras

  • PCR Purification of previous Fusion PCR
  • Digestion with NcoI/NdeI (NEBuffer4) and NcoI/KpnI (NEBuffer1 + BSA). 1 h @ 37 °C
  • Gel extraction, eluted in 30 µl water
  • Ligation (Insert:Vector 5 µl:5 µl) and transformation into DH5alpha competent cells

Friday, 09/12/2008

nothing to report


Go to 7th week

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