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

From 2008.igem.org

(Difference between revisions)
(New page: <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....)
 
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<html>
<html>
-
<link rel='stylesheet' href='http://igem-heidelberg.de/fileadmin/Wiki/Heidelberg2.css' type='text/css' />
+
 
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<link rel="stylesheet" href="http://igem-heidelberg.de/fileadmin/Wiki/Menu.css" type="text/css"/>
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<link rel="stylesheet"  href="http://igem-heidelberg.de/fileadmin/Wiki/MenuHD.css" type="text/css"/>
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<style>
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p {text-align: justify;}
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a:hover works in IE6 and FF2.
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a:hover breaks li:hover in FF2.
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div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a,
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  left: 13em; /* Places the side menu to the right of the drop-down menu.
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                            Keep it sync with the drop-down menu width.
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                            Use MenuTailor.css to customize. */
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div.MenuBar ul li ul.DropDownMenu li ul.SideMenu li a span {
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                            Keep it sync with the drop-down menu width.
 +
                            Use MenuTailor.css to customize. */
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* html div.MenuBar ul li a {
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  float: left; /* Required for IE55 and IE6.
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                            Breaks O9.
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* html ul.DropDownMenu li a:hover {
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ul.DropDownMenu li a:hover {
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* html div.MenuBar a:hover {
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* html div.MenuBar ul li table,
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* html div.MenuBar ul li table td {
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  background-color: Menu; /* Side menu unselected items.
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                            Sets the side menu "effective background" color. */
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  color: MenuText;
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div.MenuBar ul li:hover ul.DropDownMenu li:hover ul.SideMenu li a:hover,
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/*Script-Free 3-Level Menu 1.2 Tailor
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  www.CesarDaniel.info
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div.MenuBar {
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div.MenuBar ul {
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  font: 13px arial, helvetica, sans-serif; /* Required for IE55. */
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Menu Bar 1
 +
Drop-Down Menu #2
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Menu Bar 1
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Drop-Down Menu #2
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Side Menu #1
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
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div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1,
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Drop-Down Menu #2
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Side Menu #2
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,
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Drop-Down Menu #2
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Side Menu #3
 +
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 +
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 +
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 +
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,
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3 li a,
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 +
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 +
 
<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, 10/06/2008 ==
== Monday, 10/06/2008 ==
 +
* Mutagenesis PCR of LuxS,P with Phusion and the following programme (20 cycles)
 +
* PCR programme: 30 sec @ 98°C || 10 sec @ 98 °C | 30 sec @ 55 °C | 30 sec @ 72 °C || 5 min @ 72 °C | 4°C
 +
--> No PCR product
== Tuesday, 10/07/2008 ==
== Tuesday, 10/07/2008 ==
 +
* Repetition of Mutagenesis PCR with Phusion and same programme, but 30 cycles
 +
--> only LuxS2 positive --> Gelextraction
 +
 +
 +
*New PCR with Turbo Pfu and iProof (18 cycles)
 +
** 5(10) µl buffer
 +
** 1 µl forward Primer (10 µM stock)
 +
** 1 µl reverse Primer
 +
** 1 µl dNTPs
 +
** 2 µl Template
 +
** 39(34) µl water
 +
** 1 µl Polymerase
 +
** 30 sec @ 95 °C ||30 sec @ 95 °C | 60 sec @ 55 °C | 30 sec @ 68 °C || 8 min @ 68 °C | 4°C hold
== Wednesday, 10/08/2008 ==
== Wednesday, 10/08/2008 ==
 +
* Miniprep of LuxS
 +
* New PCR of LuxS and LuxP with Cloned Pfu and Phusion Polymerase
 +
** Cloned Pfu:<br/>
 +
** 30s @ 95°C || 30s @ 95°C | 1min @ 55°C | 1min @ 68°C || 8min @ 72°C | 4min (20 cycles)
 +
 +
**Phusion:<br/>
 +
**30s @ 98°C || 10s @ 98°C | 1min @ 72°C || 5min @ 72°C | 4°C (25 cycles)
 +
 +
-->NO PCR Product
 +
 +
* New PCR with Taq:
 +
** 2min @ 95°C || 30s @ 95°C | 30s @ 55°C | 1min @ 72°C || 10 min @ 72°C | 4°C (20 cycles)
 +
 +
-->NO PCR Product
 +
 +
'''Check Templates tomorrow, by digestion and undigested'''
== Thursday, 10/09/2008 ==
== Thursday, 10/09/2008 ==
 +
* Digestion of LuxS with (BamHI/NcoI and BamHI/EcoRI) and LuxP (NcoI) both negative
 +
* O/N culture of LuxS
 +
* Cloning of Tar-YFP from pDK58
 +
** Digestion of pDK58, F1-LuxP-pDK48, F2-LuxP-pDK58 with KpnI/NotI
 +
** Gelextraction eluted in 30 µl H2O
 +
** Ligation of 5µl Insert with 5µl Vector 30 min @ 30°C
 +
** Transformation into DH5a
== Friday, 10/10/2008 ==
== Friday, 10/10/2008 ==
 +
 +
[[Image:HD 081010-LuxS Digestion.png|thumb|150px|Digestion of LuxS with BamHI/EcoRI positive.]]
 +
<div style="float: right; clear: none;">[[Image:HD 081011-Fusion GA Prefix Suffix PCR.png|right|thumb|150px|Prefix/Suffix PCR of GeneArt Fusion constructs]]</div>
 +
 +
* LuxS digestion with BamHI/EcoRI (NEBuffer EcoRI + BSA) to check for insert
 +
* LuxS mutagenesis PCR for EcoRI did not work
 +
* Prefix/Suffix PCR of GeneArt Construct
 +
* PCR of LuxS1, LuxS2, LuxS3 with Phusion. Annealing Temperature Gradient 50-60°C
 +
[[Image:HD 081011-LuxS P mutagenesis prefusion.png|center|thumb|500px|PCR of LuxS and LuxP parts for mutagenesis]]
 +
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/11thweek | Go to 11th week]]
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/11thweek | Go to 11th week]]

Latest revision as of 15:39, 27 October 2008

Back to the overview

Contents

Monday, 10/06/2008

  • Mutagenesis PCR of LuxS,P with Phusion and the following programme (20 cycles)
  • PCR programme: 30 sec @ 98°C || 10 sec @ 98 °C | 30 sec @ 55 °C | 30 sec @ 72 °C || 5 min @ 72 °C | 4°C

--> No PCR product

Tuesday, 10/07/2008

  • Repetition of Mutagenesis PCR with Phusion and same programme, but 30 cycles

--> only LuxS2 positive --> Gelextraction


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

Wednesday, 10/08/2008

  • Miniprep of LuxS
  • New PCR of LuxS and LuxP with Cloned Pfu and Phusion Polymerase
    • Cloned Pfu:
    • 30s @ 95°C || 30s @ 95°C | 1min @ 55°C | 1min @ 68°C || 8min @ 72°C | 4min (20 cycles)
    • Phusion:
    • 30s @ 98°C || 10s @ 98°C | 1min @ 72°C || 5min @ 72°C | 4°C (25 cycles)

-->NO PCR Product

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

-->NO PCR Product 

Check Templates tomorrow, by digestion and undigested

Thursday, 10/09/2008

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

Friday, 10/10/2008

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


Go to 11th week

Retrieved from "http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook/10thweek"