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

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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]-->
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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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<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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                               <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>
 +
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== 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

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