Team:Heidelberg/Notebook/Sensing Group/Notebook/4thweek

From 2008.igem.org

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(→Thursday, 08/28/2008)
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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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a:hover breaks li:hover in FF2.
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* html div.MenuBar ul li a {
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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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                            Sets the side menu "effective background" color. */
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/*Script-Free 3-Level Menu 1.2 Tailor
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  www.CesarDaniel.info
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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-SM2,
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div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,
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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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<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]-->
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<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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* sequentiell and simultaneous digestion of pDk48 NcoI/NdeI and NcoI/KpnI
* sequentiell and simultaneous digestion of pDk48 NcoI/NdeI and NcoI/KpnI
--> not successful
--> not successful
-
* preparation of O/N culture of pDK48 transformed cells
+
* preparation of O/N culture for pDK48 cells
== Tuesday, 08/26/2008 ==
== Tuesday, 08/26/2008 ==
Line 132: Line 474:
=== Cloning ===
=== Cloning ===
[[Image:HD 080826-pDK48 digestions.png|right|thumb|200px|Digestion of pDK48 with xbaI and PstI yield expected results]]
[[Image:HD 080826-pDK48 digestions.png|right|thumb|200px|Digestion of pDK48 with xbaI and PstI yield expected results]]
-
* digestion of pDK48 with XbaI and PstI to check wether the vector is the right one
+
* test digestion of pDK48 with XbaI (NEBuffer 2 + BSA) and PstI (NeBuffer 3 + BSA) to check wehther the vector is the right one
-
* digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gelextraction
+
* digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gel extraction
-
* Ligation of Fusion-1 and pDk48 and transformation into DH5a competent cells
+
* Ligation of Fusion-1 fragment and pDk48 plasmid and transformation into DH5α competent cells
=== LuxS Test ===
=== LuxS Test ===
Line 151: Line 493:
== Wednesday, 08/27/2008 ==
== Wednesday, 08/27/2008 ==
* PCR of LuxQ from ''V. harveyi'' with Phusion Mastermix
* PCR of LuxQ from ''V. harveyi'' with Phusion Mastermix
-
PCR cycle: 98 °C, 5 min |98 °C, 10 sec --- 50 °C, 30 sec --- 72 °C, 1 min|30x --- 72 °C, 5 min --- 4 °C, forever
+
** PCR cycle: 5min @ 98°C || 10s @ 98°C | 30s @ 50°C | 1min @ 72°C || 5min @ 72°C | 4°C hold (30 cycles)
-
--> NO PCR product
+
** NO PCR product
-
* preparation of pTrc99a and pDK48 O/N cultures for Maxipreps
+
* preparation of pTrc99α and pDK48 O/N cultures for Maxipreps
== Thursday, 08/28/2008 ==
== Thursday, 08/28/2008 ==
-
* Maxiprep of pDK48 and pTrc99alpha (product eluted in TB buffer, not water!!)
+
* Maxiprep of pDK48 and pTrc99α (product eluted in TB buffer, not water!!)
* Digestion of LuxQ-Tar fusion (A,B) and LuxQ 1-10 with the follwing enzymes:
* Digestion of LuxQ-Tar fusion (A,B) and LuxQ 1-10 with the follwing enzymes:
*Q(1-10):
*Q(1-10):
-
*# XbaI --> if insert: 736 + 1204 bp band; if no insert: 4176 bp band  
+
*# XbaI (NEBuffer 2 + BSA) --> if insert: 736 + 1204 bp band; if no insert: 4176 bp band  
-
*# EcoRV --> if insert: 5074 bp + 866 band; if no insert: 4176 bp
+
*# EcoRV (NEBuffer 3 + BSA) --> if insert: 5074 bp + 866 band; if no insert: 4176 bp
*F1:
*F1:
-
*# BamHI --> if insert: 6101 bp band; if no insert: 5768 + 833 bp band
+
*# BamHI (NEBuffer 3 + BSA) --> if insert: 6101 bp band; if no insert: 5768 + 833 bp band
-
*# PstI --> if insert: 6101 bp; if no insert: 5768 + 833 bp band
+
*# PstI (NEBuffer 3 + BSA --> if insert: 6101 bp; if no insert: 5768 + 833 bp band
--> No result
--> No result

Latest revision as of 14:03, 29 October 2008

Back to the overview

Contents

Monday, 08/25/2008

  • sequentiell and simultaneous digestion of pDk48 NcoI/NdeI and NcoI/KpnI

--> not successful

  • preparation of O/N culture for pDK48 cells

Tuesday, 08/26/2008

Cloning

Digestion of pDK48 with xbaI and PstI yield expected results
  • test digestion of pDK48 with XbaI (NEBuffer 2 + BSA) and PstI (NeBuffer 3 + BSA) to check wehther the vector is the right one
  • digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gel extraction
  • Ligation of Fusion-1 fragment and pDk48 plasmid and transformation into DH5α competent cells

LuxS Test

Material: 500 mM IPTG-stock (A), 10 mM IPTG-stock (B)

  1. negative control: just 1 ml cell suspension
  2. 10 µM IPTG induced (1 µl stock B + 999 µl cell suspension)
  3. 20 µM IPTG induced (2 µl stock B + 998 µl cell suspension)
  4. 50 µM IPTG induced (5 µl stock B + 995 µl cell suspension)
  5. 100 µM IPTG induced (10 µl stock B + 990 µl cell suspension)
  6. 500 µM IPTG induced (1 µl stock A + 999 µl cell suspension)
  7. 1 mM IPTG induced (2 µl stock A + 998 µl celll suspension)

Measurement was done like described in the Materials & Methods section

Wednesday, 08/27/2008

  • PCR of LuxQ from V. harveyi with Phusion Mastermix
    • PCR cycle: 5min @ 98°C || 10s @ 98°C | 30s @ 50°C | 1min @ 72°C || 5min @ 72°C | 4°C hold (30 cycles)
    • NO PCR product
  • preparation of pTrc99α and pDK48 O/N cultures for Maxipreps

Thursday, 08/28/2008

  • Maxiprep of pDK48 and pTrc99α (product eluted in TB buffer, not water!!)
  • Digestion of LuxQ-Tar fusion (A,B) and LuxQ 1-10 with the follwing enzymes:
  • Q(1-10):
    1. XbaI (NEBuffer 2 + BSA) --> if insert: 736 + 1204 bp band; if no insert: 4176 bp band
    2. EcoRV (NEBuffer 3 + BSA) --> if insert: 5074 bp + 866 band; if no insert: 4176 bp
  • F1:
    1. BamHI (NEBuffer 3 + BSA) --> if insert: 6101 bp band; if no insert: 5768 + 833 bp band
    2. PstI (NEBuffer 3 + BSA --> if insert: 6101 bp; if no insert: 5768 + 833 bp band

--> No result

Friday, 08/29/2008

Go to 5th week

Retrieved from "http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook/4thweek"