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| − | <div class="kommentar">
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| − | Dieser Text kommt eher auf die Cellfree-Project site.
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| − | </div>
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| − | <h1 class="sectionedit1">Immobilizing DNA</h1> | + | <h1 class="sectionedit1">Immobilization of DNA on PDMS</h1> |
| − | <h2>Intro</h2>
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| − | <p>
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| − | One of the core components of the DiaCHIP is the copying mechanism that allows production of a protein microarray from a DNA template. The following section summarizes our achievements concerning this procedure. All together we established a <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/Cellfree_Expression" title="cellfree">cell-free expression system</a> that can be used to translate proteins from DNA immobilized on a PDMS slide. The expressed protein is then immobilized on the opposite glass slide via a specific tag system resulting in a distinct pattern. A microfluidic system is used to flush the slide with an antibody solution. Specific antibody-antigen interactions were <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Results/Diagnostics" title="diagnostics">successfully detected</a> by imaging reflectometric interference (<a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Project/iRIf" title="iRif">iRIf</a>), a label-free detection method. For diagnostic applications, the immobilized proteins are antigenic peptides specific for a certain pathogen.
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| − | </p>
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| − | <h2>DNA on PDMS</h2>
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| − | <div class="image_box left"> | + | |
| − | <div class="thumb2 trien" style="width:310px"><div class="thumbinner"><a class="lightbox_trigger" href="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_2015_freiburg_dna_on_pdms.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_2015_freiburg_dna_on_pdms.png" width="300"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=results_overview&media=2015_freiburg_dna_on_pdms.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div><strong>Figure 1: DNA immobilization on activated PDMS.</strong> PCR amplification of the expression cassette was performed with an amino-labelled reverse primer and a Cy3-labelled forward primer. Immobilized DNA is visualized by Cy3 fluorescence.</div></div></div>
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| | </div> | | </div> |
| − | <p>
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| − | The first step needed for copying the DNA array is to genetically fuse antigen coding sequences to a 10xHis tag that is used for surface immobilization later. The whole expression cassette including promoter and terminator regions is amplified by PCR using an amino-labeled reverse primer. Via this amino group, the DNA is immobilized on an activated PDMS surface. The forward primer used for this PCR is labeled with <a class=wikilink1" href="https://2015.igem.org/Team:Freiburg/Glossary" title="glossary">Cy3</a>. As it is shown in figure 1, spotting the DNA on the activated surface resulted in a distinct pattern visualized by Cy3 fluorescence.
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| | </br> | | </br> |
| | <a href="https://2015.igem.org/Team:Freiburg/Methods/Cloning">More details about vector design and cloning strategies can be found here</a>. | | <a href="https://2015.igem.org/Team:Freiburg/Methods/Cloning">More details about vector design and cloning strategies can be found here</a>. |
