Detecting and quantifying nucleic acids
Historically performed using quartz cuvettes and a UV–Vis spectrophotometer, this requires a relatively large volume of DNA (500µl – 1000µl); reading the absorbance at 260nm and 280nm enables the concentration and purity to be determined (see Application Note 116). The development of UV transparent plastics and production of micro-titre plates from these plastics has facilitated higher through put analysis using suitably equipped microplate readers . Readers are capable of DNA quantitation in different plate formats typically 96 and 384 well and for ultra low volumes in the LVis plate.
The filter based FLUOstar OPTIMA and the previous Galaxy models provide a simple 260/280 ratio (Application Note 116).
The development of the spectrometer based FLUOstar Omega provides more data per sample allowing more detailed examination on the quality of the nucleic acid sample. The FLUOstar Omega has the ability to simultaneously acquire the entire spectrum (220-1000nm) in less than 1 second per well. The ability to report 230, 260, 280, 340 nm and analyse in MARS using pre-defined templates makes high through-put DNA analysis simple. Using half area 96 well plates or 384 well plates limits the amount of sample required and as the read is non-destructive the majority of the sample can be recovered.
The launch of the SPECTROstar Nano and the associated LVis plate has provided the ability to perform the analysis of up to 16 low volume (2µl) samples.
Fluorescent probes have also successfully been used to measure DNA concentration and provide a highly robust and reproducible high through method (Application Note 103)