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

From 2008.igem.org

(Difference between revisions)
(→Thursday, 08/28/2008)
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<body>
<body>
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</li>
</li>
<li>
<li>
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<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">
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<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]-->
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<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">
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<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]-->
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<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">
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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]-->

Revision as of 15:36, 27 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 DH5alpha 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 pTrc99a and pDK48 O/N cultures for Maxipreps

Thursday, 08/28/2008

  • Maxiprep of pDK48 and pTrc99alpha (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"