Abstract
The use of the microliter measurement cell μCuvette, in combination with the BioPhotometer or BioSpectrometer, enables determination of high concentrations of nucleic acids or proteins using minimum volumes. In order to achieve optimum results, the measurement principle will be elaborated in this application note, and use and handling of the μCuvette are described in detail. Possible factors which may influence measurements, as well as individual instrument parameters, will also be discussed. In addition, comparative measurements with other cuvettes and microvolume cuvettes were performed, with a focus on self absorption, precision and accuracy.
Introduction
Following isolation of nucleic acids or proteins, it is usually necessary to determine the concentration of these biomolecules photometrically in order to prepare them for downstream applications. Since the samples are often highly concentrated following isolation, dilution is frequently required. In order to avoid faulty calculation of the originating concentration, and thus faulty calculation for subsequent applications, the individual dilution steps need to be executed with extreme accuracy. The challenge lies in the fact that most samples become unusable following dilution; renewed concentration of the samples would prove rather labor-intensive. In order to avoid such serial dilutions, microliter measurement systems may be used which are capable of measuring highly concentrated samples without the need for dilution. For these measurements, a few microliters suffice. This is made possible by the shortened light path employed by the microliter measurement systems. As shown in figure 1, when a certain sample concentration is reached in a normal cuvette featuring a 10 mm light path, light is unable to pass through the solution and therefore cannot reach the detector within the photometer. In the case of a microliter measurement cell with abbreviated path length, the path is not long enough to block the light from passing through the very same sample. Therefore, the light reaches the detector, and the concentration can be determined under consideration of the abbreviated light path.
Part 1: Handling of the μCuvette
In order to implement the measurement principle depicted in figure 1, Eppendorf developed the μCuvette (figure 2). The μCuvette consists of two arms; at the end of each resides a small quartz plate. To facilitate sample application onto the sample application area, two black markings can be found at the end of both arms.
Figure 1: Abbreviation of the light path for photometric determination of highly concentrated samples (not to scale): A) Standard cuvette B) Microliter measurement cell While the sample concentration may not be determined in a cuvette with a path length of 10 mm, the sample can be measured in a cuvette with a path length of 1 mm.
Figure 2: The μCuvette: A: Sample application area and optical measurement window B: Quartz plates C: Spacer>> Download the full Application Note as PDF
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