Biomod/2011/LMU/FolD'N'Assemble/Methods
Cadnano
The design of the nanopill was made using the CAD software caDNAno. Cadnano allows an easy design of DNA structures, by offering an accessible interface for keeping track of the scaffold path through the 3D structure and assisting the user with the generation and adjustment of complimentary DNA staples. A very good tutorial with further information and examples is avalaible at
Gel electrophoresis
Gel electrophoresis is an established method to sort molecules by size and charge. The basic principle is to position the samples into wells inside a gel block and to apply voltage to the gel. The charged molecules migrate in the electric field, but due to the matrix structure of the gel, the different sized molecules all travel with varying velocity. After some time, distinct bands of same-velocity molecules form in the gel and can be extracted or analyzed by different methods. Gel electrophoresis can also be used for the purpose of filtration and purification.
For all of the experiments, a 2% agarose gel with TBE (Tris borate EDTA) as running buffer was used. Mg2+ is added in the form of MgCl2 to reduce the repulsion of the closely packed negative DNA backbones and to stabilize the DNA structures. A voltage of 70 V is then applied to the gel block for 3 hours. Afterwards the DNA in the gel is made visible by staining with ethidium bromide. Ethidium bromide (EtBr) intercalates DNA and emits florescence light if excited by UV radiation. DNA that came into contact with EtBr can therefore be visualized by using an UV lamp.
TEM
TEM(Transmission Electron Microscope) is a form of microscopy that relies on electrons, instead of light, to image samples. The electrons interact with the sample and the interaction is imaged by CCD sensors. The advantage compared to light microscopy is that electrons have a relatively low de-broglie wavelength, this makes resolution of objects possible that can not be imaged by light microscopy. The samples are first stained with uranyl acetate, it attaches to the DNA structures and interferes with the electrons. This interaction creates negative images of the structures on the CCD sensor.
