Absorbance or fluorescence?

The most common methods for DNA quantification are UV spectrophotometry and fluorescence measurements 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. This is the first decision before choosing an instrument for the DNA quantification method to be used. Learn more about DNA quantification methods.

Absorbance readers

The two most important options to quantify DNA concentrations by absorption measurements are microvolume spectrophotometers and absorbance microplate readers.

Microvolume Spectrophotometer

In the past, the cuvette spectrophotometer was the only available option for quantifying DNA concentration by absorption measurements. This application was very limited due to the large sample volume required and the small sample volume available in molecular biology. The sensitivity of a cuvette spectrophotometer is better than that of a microvolume spectrophotometer, but the required sample volume 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 allows the measurement of tiny drops of the sample of typically 1 µL. The need for cuvette spectrophotometers for DNA quantification has been largely abandoned 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. This is rather time consuming when large numbers of samples need to be quantified and the use of a microplate reader is needed to measure more samples in less time.

Microplate absorbance readers

Microplate readers can measure many samples in a short time and the typical plate formats are 96- and 384-well but some readers can also read plates with 1536 wells or more. Microplate readers do have some limitations in calculating DNA concentrations compared to microvolume spectrophotometers:

  • Microplate readers require larger sample volumes for the measurement, but this can depend on the plate type and manufacturer. The higher the well density the lower the minimum working volume, and there are low-volume versions of common microplate formats available. The 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 and special microplates that do not block 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 is an important factor when calculating DNA concentration (see DNA quantification methods) and depends on well geometry and sample volume. The formula to calculate DNA concentration requires a pathlength of 1 cm and pathlength correction must 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 µDrop plate, 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 purity of DNA). Both filter-based and monochromator-based readers are suitable for this application.