Describe how ultra-fast, full spectrum DNA measurements are performed on the SPECTROstar Nano in three different volume formats: microplates, cuvettes, and low volume samples.
Most substances in solution absorb light at a specific wavelength. When a defined amount of light is sent through a solution, a certain amount of light is absorbed by the substance. There is a direct linear relation between this absorbed light and the concentration of the solute (up to certain limits). This relation is shown in the Beer-Lambert law: A = b · c · ε, where b = pathlength [cm]; c = concentration of solute [mol/l or M]; and ε = substance-specific extinction coefficient [cm-1 M-1]. The reciprocal value of the coefficient at a 1 cm pathlength can be used to determine the concentration of nucleic acids without preparing a standard curve. Reciprocal extinction coefficients for dsDNA, ssDNA, and for RNA are widely known (50, 33, and 40 μg/mL, respectively).
The pathlength needs to be normalized to 1 cm in order to be used in the Beer-Lambert law. With the SPECTROstar Nano, cuvettes, microplates, and low volume samples (2 μl) can all be measured. Cuvette measurements are automatically normalized to the 1 cm length of the cuvette. In microplates the pathlength will vary depending on the liquid volume and well size. This can be normalized to a 1 cm pathlength by either: a) using a microplate with a defined pathlength and volume (the low volume LVis Plate has a pathlength of 0.5 mm when using 2 μL); b) using a pathlength correction where the volume and microplate dimensions are used in a mathematical algorithm; or c) using a known water peak value correction to normalize the data. With the SPECTROstar Nano, all three methods can be done.
Purified bacteria plasmid DNA was measured on the SPECTROstar Nano using a 1 cm cuvette, BMG LABTECH’s LVis plate, and two microplates (96- and 384-well). The figure shows a full spectrum measurement of different concentrations of dsDNA in a 384-well microplate. Full spectrum analysis and BMG LABTECH’s MARS data analysis software quickly and easily determines the DNA peak (260 nm), as well as the water peak (980 nm, not shown) and impurities at other wavelengths (230, 280, and 340 nm).
DNA samples measured in cuvettes, low volumes (LVis Plate), and microplates (96 and 384-well) all gave similar and reproducible results. Purity of DNA samples was determined using other wavelengths: protein (280 nm), phenolate or thiocyanate (230 nm), and cell particulates (340 nm).
Measuring DNA samples with the SPECTROstar Nano can be done in different formats: cuvettes, standard microplates, and low volumes using the LVis Plate. If there is enough DNA and only a few samples will be measured, cuvette measurements can be performed. For higher throughput a microplate can be used, or if sample volume is very limited, then the LVis Plate is recommended.
In addition, ultra-fast, full spectrum analysis allows for the samples to be measured only once. This greatly reduces reading times and errors due to measuring multiple times for multiple wavelengths.
Figure Legend: Full spectrum analysis on the SPECTROstar Nano is used to quickly measure DNA (260 nm) as well as impurities such as protein (280 nm), phenolate or thicocyanate (230 nm), and cell particulates (340 nm).