Team:Heidelberg/Notebook/Sensing Group/Notebook/9thweek

From 2008.igem.org

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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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<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]-->
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<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, 09/30/2008 ==
== Tuesday, 09/30/2008 ==
* Miniprep of LuxS+pDK6 and F1/F2-Luxp-pBAD33
* Miniprep of LuxS+pDK6 and F1/F2-Luxp-pBAD33
-
* Digestion of GeneArt Fusion constructs with EcoRI/PstI and Gel-Extraction  
+
* Digestion of GeneArt Fusion constructs with EcoRI/PstI (NEBuffer EcoRI + BSA) and Gel-Extraction  
-
* Digestion of GeneArt Fusion constructs with EcoRI/PstI and Gel-Extraction
+
* Digestion of GeneArt Fusion constructs with EcoRI/PstI (NEBuffer EcoRI + BSA) and Gel-Extraction
-
== Wednesday, 09/29/2008 ==
+
== Wednesday, 10/01/2008 ==
 +
* Miniprep of Fusion-LuxP-pBAD33 (both constructs)
 +
* Digestion of pSB1A3 with EcoRI,PstI,NdeI (3µl DNA, 40 µl total volume)
 +
* Digestion of pSB2K3 (10µl DNA, 20µl total volume) with EcoRI/PstI (NEBuffer EcoRI + BSA)
 +
[[Image:HD 081001-F-PCR prefix speI.png|center|thumb|400px|PCR GeneArt Fusion constructs with Prefix/Suffix primer yielded no product. F1/F2 with prefix/speI primer were positive, but mutation strategy dropped later. A mix of LuxS and pDK6 was added on the gel to check if double transformation was successfull. Digestions of pSB1A3 and pSB2K3 with EcoRI/Pst were supposed to be used for subsequent standardization of parts]]
-
== Thursday, 09/30/2008 ==
+
== Thursday, 10/02/2008 ==
 +
* PCR like yesterday overnight, but with 42°C annealing and as template with EcoRI/PstI digested Fusion constructs
 +
* Maxiprep of GeneArt Fusion Constructs and pSB2K3
 +
* PCR Purification of F1/F2 Prefix/SpeI PCR products
 +
* Digestion of F1-prefix/SpeI and F2-prefix/SpeI with EcoRI/SpeI (NEBuffer EcoRI + BSA,610 bp band)
 +
* Digestion of F1/F2-LuxP-pDK48 with EcoRI,NotI,XbaI
 +
** 4686 bp
 +
** 1330 bp (wanted band)
 +
** 599 bp
 +
** 587 bp
 +
* Mutagenesis PCR of LuxS (LuxSmutF/LuxSmutR) and LuxP (LuxPmutF/LuxPmutR) overnight
-
== Friday, 10/01/2008 ==
+
== Friday, 10/03/2008 ==
 +
* Digestion of mutagenesis PCR products with DpnI, 1h @ 37 °C
 +
--> Analysis on 1% Agarose-Gel --> No band visible
 +
* Ligation of F1/2 (GA) with pSB1A3 and pSB2K3, 1h @ RT
 +
--> Transformation into DH5a. Plated over weekend.
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/10thweek | Go to 10th week]]
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/10thweek | Go to 10th week]]

Latest revision as of 15:39, 27 October 2008

Back to the overview


Contents

Tuesday, 09/30/2008

  • Miniprep of LuxS+pDK6 and F1/F2-Luxp-pBAD33
  • Digestion of GeneArt Fusion constructs with EcoRI/PstI (NEBuffer EcoRI + BSA) and Gel-Extraction
  • Digestion of GeneArt Fusion constructs with EcoRI/PstI (NEBuffer EcoRI + BSA) and Gel-Extraction

Wednesday, 10/01/2008

  • Miniprep of Fusion-LuxP-pBAD33 (both constructs)
  • Digestion of pSB1A3 with EcoRI,PstI,NdeI (3µl DNA, 40 µl total volume)
  • Digestion of pSB2K3 (10µl DNA, 20µl total volume) with EcoRI/PstI (NEBuffer EcoRI + BSA)
PCR GeneArt Fusion constructs with Prefix/Suffix primer yielded no product. F1/F2 with prefix/speI primer were positive, but mutation strategy dropped later. A mix of LuxS and pDK6 was added on the gel to check if double transformation was successfull. Digestions of pSB1A3 and pSB2K3 with EcoRI/Pst were supposed to be used for subsequent standardization of parts

Thursday, 10/02/2008

  • PCR like yesterday overnight, but with 42°C annealing and as template with EcoRI/PstI digested Fusion constructs
  • Maxiprep of GeneArt Fusion Constructs and pSB2K3
  • PCR Purification of F1/F2 Prefix/SpeI PCR products
  • Digestion of F1-prefix/SpeI and F2-prefix/SpeI with EcoRI/SpeI (NEBuffer EcoRI + BSA,610 bp band)
  • Digestion of F1/F2-LuxP-pDK48 with EcoRI,NotI,XbaI
    • 4686 bp
    • 1330 bp (wanted band)
    • 599 bp
    • 587 bp
  • Mutagenesis PCR of LuxS (LuxSmutF/LuxSmutR) and LuxP (LuxPmutF/LuxPmutR) overnight

Friday, 10/03/2008

  • Digestion of mutagenesis PCR products with DpnI, 1h @ 37 °C

--> Analysis on 1% Agarose-Gel --> No band visible

  • Ligation of F1/2 (GA) with pSB1A3 and pSB2K3, 1h @ RT

--> Transformation into DH5a. Plated over weekend.

Go to 10th week

Retrieved from "http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook/9thweek"