Difference between revisions of "Team:Aachen/Wetlab/Workflow"

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<h1>Wetlab</h1>
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<h1>Workflow</h1>
 
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<h2>Overview</h2>
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<p>Each good project needs a good organisation and strategy. For a better understanding about what and why we did in our laboratory, we created this flowchart with a detailed overview.</p>
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<p>Our project is based on specific receptors for melatonin, that we express in S. cerevisiae for whole-cell based assay and in E. coli for the cell-free hardware. <br></br>
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If you would like to get an overview of the work we did in the wetlab and the protocols we used, just click on the icons below. <br>
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You can also find information about our participation in the Interlab Studies, as well as our biobrick here.</p>
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<p>First of all we planned which genes and homologue regions we wanted to use and we designed their respective primers using <a href= "https://www.geneious.com"> Geneious </a>. For this process we used always this common pattern</p>
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<img class="img-fluid" src="https://static.igem.org/mediawiki/2018/d/d5/T--Aachen--Workflow-02.png">
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<a href="https://2018.igem.org/Team:Aachen/Wetlab/Parts"><img src="https://static.igem.org/mediawiki/2018/c/c0/T--Aachen--parts-icon.png" alt="Part" style="max-width:250px; height=auto; padding-right: 100px;"></a>
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<p>The most important characteristic of the designed primers for the production of the DNA Fragments was the use of overhangs. With this primers with overhangs we generate the desired fragments with homologous parts.</p>
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<a href="https://2018.igem.org/Team:Aachen/Wetlab/Notebook"><img src="https://static.igem.org/mediawiki/2018/8/83/T--Aachen--labbook-icon.png" alt="Labbook" style="max-width:250px; height=auto; padding-right: 100px;"></a>
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<img class="img-fluid" src="https://static.igem.org/mediawiki/2018/e/e3/T--Aachen--Workflow-06.png">
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<a href="https://2018.igem.org/Team:Aachen/Wetlab/Protocols"><img src="https://static.igem.org/mediawiki/2018/7/7a/T--Aachen--protocols-icon.png" alt="Protocols" style="max-width:250px; height=auto; padding-right: 100px;"></a>
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<img class="img-fluid" src="https://static.igem.org/mediawiki/2018/d/d5/T--Aachen--Workflow-10.png">
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<a href="https://2018.igem.org/Team:Aachen/InterLab"><img src="https://static.igem.org/mediawiki/2018/a/ab/T--Aachen--interlab-icon.png" alt="Interlab" style="max-width:250px; height=auto; padding-right: 100px;"></a>
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<p>After the production of all desired fragments we assembled them using the <a href= "https://www.neb.com/products/e5520-nebuilder-hifi-dna-assembly-cloning-kit#Product%20Information"> Assembly Kit </a> from New England Biolabs. Afterwards we verificated the assembly product with PCRs, electrophoresis and sequencing. </p>
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<p>With the already good assembled fragments we transformed the yeast strains using the <a href= "https://www.zymoresearch.de/frozen-ez-yeast-transformation-ii-kittm"> Frozen-EZ Yeast Transformation II Kit </a> from Zymo Research. </p>
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<img class="img-fluid" src="https://static.igem.org/mediawiki/2018/8/88/T--Aachen--asfkn.png">
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<p>To review the transformed yeast we isolated their genome with the <a href= "https://2018.igem.org/Team:Aachen/Wetlab/Protocol19"> Quick and Dirty protocol </a>and after a positive verification-PCR with the respective primers, the sequencing of the introduced gene was given in order to Eurofins.</p>
 
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Revision as of 02:53, 18 October 2018

Workflow

Each good project needs a good organisation and strategy. For a better understanding about what and why we did in our laboratory, we created this flowchart with a detailed overview.







First of all we planned which genes and homologue regions we wanted to use and we designed their respective primers using Geneious . For this process we used always this common pattern



The most important characteristic of the designed primers for the production of the DNA Fragments was the use of overhangs. With this primers with overhangs we generate the desired fragments with homologous parts.




After the production of all desired fragments we assembled them using the Assembly Kit from New England Biolabs. Afterwards we verificated the assembly product with PCRs, electrophoresis and sequencing.

With the already good assembled fragments we transformed the yeast strains using the Frozen-EZ Yeast Transformation II Kit from Zymo Research.



To review the transformed yeast we isolated their genome with the Quick and Dirty protocol and after a positive verification-PCR with the respective primers, the sequencing of the introduced gene was given in order to Eurofins.