Team:Heidelberg/Notebook/Sensing Group/Notebook/8thweek

From 2008.igem.org

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(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>
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<link rel='stylesheet' href='http://igem-heidelberg.de/fileadmin/Wiki/Heidelberg2.css' type='text/css' />
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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]-->
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<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]-->
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<ul class="DropDownMenu" id="MB1-DDM3">
<ul class="DropDownMenu" id="MB1-DDM3">
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</li>
</li>
<li>
<li>
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<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]-->
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<ul class="DropDownMenu" id="MB1-DDM5">
<ul class="DropDownMenu" id="MB1-DDM5">
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</li>
<li style="width: 160px">
<li style="width: 160px">
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<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]-->
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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]-->
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== Monday, 09/22/2008 ==
== Monday, 09/22/2008 ==
 +
* test digestion of F1_LuxP_NdeI (F1 cloned in pDK48 with NdeI), F2_LuxP_NdeI, F1_LuxP_KpnI (F1 cloned in pDK48 with KpnI) and F2_LuxP_KpnI (F2 cloned in pDK48 with KpnI) with NcoI (NEBuffer 3)
 +
[[Image:HD 080922 Fusion LuxP in pDK48.png|center|thumb|400px|Digestions of Fusion constructs with NcoI. Positive clones show two bands at 4308 and 2915 bp]]
 +
* Transformation of Fusion constructs (F1(NdeI)_LuxP_pDK48, F1(KpnI)_LuxP_pDK48 and F2(NdeI)_LuxP_pDK48) into MG1655 and HCB33 cells
 +
* Preparation of cultures for Maxipreps of Fusion constructs
== Tuesday, 09/23/2008 ==
== Tuesday, 09/23/2008 ==
 +
* Maxiprep of LuxP-Fusion constructs (F1(NdeI)_LuxP_pDK48, F1(KpnI)_LuxP_pDK48 and F2(NdeI)_LuxP_pDK48)
 +
* Cloning of Fusion_LuxP in pBAD33 with BamHI/PstI
 +
* digestions of Fusion constructs and pBAD33 as described in gel image descriptions
 +
[[Image:HD 080923 Fusion-LuxP pBAD33 Digestion.png|left|thumb|200px|Digestion of Fusion constructs and pBAD33 with BamHI/PstI for subsequent cloning.]][[Image:HD 080923 Fusion-LuxP Digestion.png|left|thumb|400px|Digestion of Fusion constructs with NcoI and PstI to check for correct insert and Maxiprep digetions.]]
 +
 +
<br style="clear: both;"/>
 +
 +
* O/N culture of Fusion-LuxP-pDK48 in HCB33 and MG1655 in TB-Kan
 +
* Transformation of Fusion-LuxP-pBAD33 constructs into DH5a
== Wednesday, 09/24/2008 ==
== Wednesday, 09/24/2008 ==
 +
* Glycerol-Stock of Fusion1-LuxP-pDK48 and Fusion2-LuxP-pDK48
 +
* O/N culture of Fusion-LuxP-pDK48 in HCB33 and MG1655 in LB
 +
* O/N culture of LuxS and GFP Plasmid from Marcus
 +
* Re-Sequencing of Fusion-1 and Fusion-2
 +
Correct sequence for both Fusion constructs confirmed
== Thursday, 09/25/2008 ==
== Thursday, 09/25/2008 ==
 +
* Miniprep of Fusion-LuxP-pDK48 constructs, LuxS, GFP Plasmid from Marcus
 +
* Competent Cells (Top-10, UU1250)
 +
* Picked colonies of Fusion-LuxP-pBAD33
== Friday, 09/26/2008 ==
== Friday, 09/26/2008 ==
 +
* Miniprep of Fusion-LuxP-pBAD33 constructs and Digestion with HindIII (NEBuffer2)
 +
** 5 µl DNA
 +
** 1µl NEBuffer 2
 +
** 0.5µl HindIII
 +
** 3.5µl H<sub>2</sub>O
 +
* Expected Fragments
 +
** Fusion1-LuxP-pBAD33: 7616bp and 769bp
 +
** Fusion2-LuxP-pBAD33: 7631bp and 769bp
 +
** pBAD33 only: 5360bp
 +
[[Image:HD 080926 F LuxP pBAD digestion.png|center|thumb|500px|Double digestion of Fusion constructs in pBAD33 with BamHI/PstI and single digestion with HindIII. Double digestion yielded positive results for all constructs.]]
 +
* Double Transformations of Fusion-LuxP-pBAD33 and pDK4 into UU1250 (2 Amp-CM plates) as well as LuxS and pDK6 into DH5a (Amp-Kan plate(s))
 +
* Transformation of pDK4 into DH5a (CM plate)
 +
* Preparation of 2x 3mL LB-CM-Amp, 2x 5mL TB-CM-Amp, 1x 3mL LB-Amp-Kan, 1x 5mL TB-Amp-Kan, 1x 5mL LB-CM
 +
 +
 +
== Sunday, 09/28/2008 ==
 +
* Picked colony of each Fusion-LuxP-pBad/pDK4 UU1250 cells and inoculated in 5 mL TB and 5 mL LB medium
 +
* No colonies on LuxS/pDK6 DH5a cell plate. 5 mL liquid culture of LuxS DH5a cells.
 +
* No colonies on pDK4 cell plate. Waiting until tomorrow
 +
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/9thweek | Go to 9th week]]
[[Team:Heidelberg/Notebook/Sensing_Group/Notebook/9thweek | Go to 9th week]]

Latest revision as of 15:38, 27 October 2008

Back to the overview

Contents

Monday, 09/22/2008

  • test digestion of F1_LuxP_NdeI (F1 cloned in pDK48 with NdeI), F2_LuxP_NdeI, F1_LuxP_KpnI (F1 cloned in pDK48 with KpnI) and F2_LuxP_KpnI (F2 cloned in pDK48 with KpnI) with NcoI (NEBuffer 3)
Digestions of Fusion constructs with NcoI. Positive clones show two bands at 4308 and 2915 bp
  • Transformation of Fusion constructs (F1(NdeI)_LuxP_pDK48, F1(KpnI)_LuxP_pDK48 and F2(NdeI)_LuxP_pDK48) into MG1655 and HCB33 cells
  • Preparation of cultures for Maxipreps of Fusion constructs

Tuesday, 09/23/2008

  • Maxiprep of LuxP-Fusion constructs (F1(NdeI)_LuxP_pDK48, F1(KpnI)_LuxP_pDK48 and F2(NdeI)_LuxP_pDK48)
  • Cloning of Fusion_LuxP in pBAD33 with BamHI/PstI
  • digestions of Fusion constructs and pBAD33 as described in gel image descriptions
Digestion of Fusion constructs and pBAD33 with BamHI/PstI for subsequent cloning.
Digestion of Fusion constructs with NcoI and PstI to check for correct insert and Maxiprep digetions.


  • O/N culture of Fusion-LuxP-pDK48 in HCB33 and MG1655 in TB-Kan
  • Transformation of Fusion-LuxP-pBAD33 constructs into DH5a

Wednesday, 09/24/2008

  • Glycerol-Stock of Fusion1-LuxP-pDK48 and Fusion2-LuxP-pDK48
  • O/N culture of Fusion-LuxP-pDK48 in HCB33 and MG1655 in LB
  • O/N culture of LuxS and GFP Plasmid from Marcus
  • Re-Sequencing of Fusion-1 and Fusion-2 
Correct sequence for both Fusion constructs confirmed

Thursday, 09/25/2008

  • Miniprep of Fusion-LuxP-pDK48 constructs, LuxS, GFP Plasmid from Marcus
  • Competent Cells (Top-10, UU1250)
  • Picked colonies of Fusion-LuxP-pBAD33

Friday, 09/26/2008

  • Miniprep of Fusion-LuxP-pBAD33 constructs and Digestion with HindIII (NEBuffer2)
    • 5 µl DNA
    • 1µl NEBuffer 2
    • 0.5µl HindIII
    • 3.5µl H2O
  • Expected Fragments
    • Fusion1-LuxP-pBAD33: 7616bp and 769bp
    • Fusion2-LuxP-pBAD33: 7631bp and 769bp
    • pBAD33 only: 5360bp
Double digestion of Fusion constructs in pBAD33 with BamHI/PstI and single digestion with HindIII. Double digestion yielded positive results for all constructs.
  • Double Transformations of Fusion-LuxP-pBAD33 and pDK4 into UU1250 (2 Amp-CM plates) as well as LuxS and pDK6 into DH5a (Amp-Kan plate(s))
  • Transformation of pDK4 into DH5a (CM plate)
  • Preparation of 2x 3mL LB-CM-Amp, 2x 5mL TB-CM-Amp, 1x 3mL LB-Amp-Kan, 1x 5mL TB-Amp-Kan, 1x 5mL LB-CM


Sunday, 09/28/2008

  • Picked colony of each Fusion-LuxP-pBad/pDK4 UU1250 cells and inoculated in 5 mL TB and 5 mL LB medium
  • No colonies on LuxS/pDK6 DH5a cell plate. 5 mL liquid culture of LuxS DH5a cells.
  • No colonies on pDK4 cell plate. Waiting until tomorrow


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