Absorbance or fluorescence?
The most common methods forare UV spectrophotometry and fluorescence measurement of DNA-binding dyes. The first method requires a measuring system that is suitable for absorption measurement and the last requires a device that can be used for fluorescence measurement. Therefore, the first decision before choosing an instrument is the DNA quantification method to be used. .
The two most important options or quantifying DNA concentration by absorption measurements areand .
In the past, the cuvette spectrophotometer was the only available option for quantifying DNA concentration by absorption measurements. However, its application was very limited due to the large sample volume required and the small available sample volume in molecular biology. Although the sensitivity of a cuvette spectrophotometer is better than that of a, the required sample quantity is very large: 300-400 µL in semi-micro cuvettes and 70 µL in ultra-micro cuvettes. A real breakthrough in the application of the method was only achieved with the introduction of microvolume spectrophotometers, which allow the measurement of tiny drops of the sample (typically 1 µL). Therefore, cuvette spectrophotometers for DNA quantification have been largely abandoned in the meantime, and microvolume spectrometers are the instrument of choice for single sample absorption measurements.
Quantifying DNA with a microvolume spectrophotometer is simple and straightforward, but samples must be measured individually with a short cleaning step between samples, which is rather time consuming when large numbers of samples need to be quantified. Therefore, the use of a microplate reader is recommended to measure more samples in less time.
Microplate absorbance readers
can measure many samples in a short time: Typical plate formats are 96- and 384-well, but some readers can also read plates with 1536 wells or more. However, they have some limitations in calculating DNA concentrations compared to microvolume spectrophotometers:
- They require larger sample volumes for the measurement. This depends on the plate type and manufacturer. The higher the well density, the lower the minimum working volume, and there are low-volume versions of the most common microplate formats available. In most cases, however, measurement in microplates still requires several times the sample volume compared to measurement in a microvolume spectrophotometer. In the table at the end of this article you will find the minimum working volume of common microplate formats.
- Standard polystyrene microplates cannot be used because they block UV light. Special microplates that are transparent to UV light are currently available from most major manufacturers. Although they are more expensive than polystyrene microplates, they are more affordable and convenient than quartz or quartz-bottom microplates.
- Pathlength, which is a factor in the formula to calculate DNA concentration (see ) depends on well geometry and sample volume. As the formula to calculate DNA concentration requires a pathlength of 1 cm, pathlength correction has to be used in microplate measurements. Pathlength correction can be easily calculated by measuring absorbance of the samples at 900 and 975 nm.
Microvolume microplates for DNA quantification
A workaround for all three problems is the use of microvolume microplates for DNA quantification. This type of microplates uses small sample volumes (usually 2 µL), is UV-transparent, and has a fixed pathlength that can be easily applied to the calculations without additional measurements. While they don’t have as many sample positions as standard microplates (normally 16 instead of 96), they offer a good compromise between sample volume and throughput. For convenient quantification of DNA in our microplate readers, we offer the, including parameter files for easy measurement and calculation using the MikroWin software.
As far as the choice of microplate reader is concerned, the only requirement is to be able to measure absorbance in the UV, down to 230 nm (as this wavelength is used to assess the). Both filter-based and monochromator-based readers are suitable for this application.