Difference between revisions of "Team:Freiburg/Results/Immobilization"
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<div class="level1"> | <div class="level1"> | ||
</div> | </div> | ||
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+ | <h3 class="sectionedit2">PDMS surface</h3> | ||
+ | |||
+ | Before the DNA can be spotted onto the PDMS slide, the PDMS surface has to be prepared in a specialized manner (link protocol). | ||
+ | To allow binding of a amio-tagged DNA a PDITC layer is coated (?) on top of the PDMS slide. | ||
+ | (Bild: layers) | ||
+ | |||
+ | <h3 class="sectionedit2">PCR with Cy3-labelled primer and amino-labelled primer</h3> | ||
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+ | To bind the DNA on the PDMS surface it has to be fused to a amino tag on the 3'-end. The amino-tag can interact with the PDITC-PDMS surface. | ||
+ | </br> | ||
+ | A Cy3-label is fused on the 5'-end of the DNA, thereby allowing detection of the spotted DNA via Cy3 measuring. | ||
+ | PCR amplification of the coding sequence of GFP with the above mentioned primers was succesful. To verify correct DNA amplification an analysis on an agarose-gel was performed (fig.1). | ||
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+ | <h3 class="sectionedit2">Spotting of DNA - measuring of Cy3 signal</h3> | ||
+ | |||
+ | DNA was spotted onto the prepared PDMS slide with a concentration of 25ng/µl. The slide was subsequently incubated over night and the DNA-solution was dried afterwards at 60°C. After washing of the slide (?) the DNA could be detected on the slide via measuring of the Cy3 signal. | ||
+ | </br> | ||
+ | Bild: Cy3-DNA on PDMS | ||
+ | </br> | ||
+ | |||
+ | <h3 class="sectionedit2">Cell-free expression of GFP from spotted DNA</h3> | ||
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+ | A second method to verify binding of the DNA to the PDMS slide is cell-free expression of the DNA in a microfluidic chamber. DNA was spotted on the PDMS slide as described above and cell-free expression mix was flushed into the chamber. The slide was incubated with the cell-free expression mix for 2 hours, during which GFP was expressed. | ||
+ | We could show successful expression of GFP from the spotted DNA using a fluorescence microscope. | ||
+ | </br> | ||
+ | Bild: GFP fluorescence | ||
+ | </br> | ||
Text | Text | ||
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<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>. | ||
</p> | </p> | ||
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</div> | </div> | ||
Revision as of 18:59, 13 September 2015