Sizing of single fluorescently stained DNA fragments by scanning microscopy

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO‐1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly‐l‐lysine, 3‐aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA‐sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7-14%. The proposed method is straightforward and can be applied to standardized microtiter plate

Sizing of single fluorescently stained DNA fragments by scanning microscopy

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-l-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7–14%. The proposed method is straightforward and can be applied to standardized microtiter plates

Terahertz Time-Domain Polarimetry in Reflection for Film Characterization

Terahertz time-domain spectroscopy is a useful technique to characterize layered samples and thin films. It gives access to their optical properties and thickness. Such measurements are done in transmission, which requires access to the sample from opposite sides. In reality this is not always possible. In such cases, reflection measurements are the only option, but they are more difficult to implement. Here we propose a method to characterize films in reflection geometry using a polarimetric approach based on the identification of Brewster angle and modeling of the measured signal to extract the refractive index and thickness of the sample. The technique is demonstrated experimentally on an unsupported single layer thin film sample. The extracted optical properties and thickness were in good agreement with established transmission terahertz spectroscopy measurements. The new method has the potential to cover a wide range of applications, both for research and industrial purposes

Understanding protein adsorption phenomena at solid surfaces

Protein adsorption at solid surfaces plays a key role in many natural processes and has therefore promoted a widespread interest in many research areas. Despite considerable progress in this field there are still widely differing and even contradictive opinions on how to explain the frequently observed phenomena such as structural rearrangements, cooperative adsorption, overshooting adsorption kinetics, or protein aggregation. In this review recent achievements and new perspectives on protein adsorption processes are comprehensively discussed. The main focus is put on commonly postulated mechanistic aspects and their translation into mathematical concepts and model descriptions. Relevant experimental and computational strategies to practically approach the field of protein adsorption mechanisms and their impact on current successes are outlined