Team:Freiburg/Results/Immobilization
Sabine kannst du das bitte korrigieren usw. Ich hab einfach mal versucht was zu schreiben, aber ich hab echt nicht so viel Plan ^^ (LS 13/9)
Immobilization of DNA on PDMS
Establishing a specific PDMS surface
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)PCR amplification of DNA with Cy3-labelled primer and amino-labelled primer
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. 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).Spotting of DNA - measuring of Cy3 signal
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. Bild: Cy3-DNA on PDMSCell-free expression of GFP from spotted DNA
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. Bild: GFP fluorescence Text More details about vector design and cloning strategies can be found here.
