Instruments for DNA quantification - Berthold Technologies GmbH & Co.KG

Absorbance or fluorescence?

The most common methods for DNA quantification are 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. Learn more about DNA quantification methods.

Absorbance readers

The two most important options or quantifying DNA concentration 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. 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 microvolume spectrophotometer, 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

Microplate 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 DNA quantification methods) 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 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.

Fluorescence Readers

If you need high sensitivity or specificity in your DNA quantification measurements, you should switch from absorbance to fluorescence, which requires a fluorometer. As with absorbance readers, cuvette fluorometers are not considered, because of the large amount of sample they require. This provides us with two basic options: compact tube fluorometers and fluorescence microplate readers.

These are single-tube fluorometers that use 0.5 mL PCR tubes as the sample format. They are often offered by manufacturers of DNA quantification kits and are therefore best suited for this manufacturer´s kits. While they´re very affordable, they can usually only measure at 2 wavelength pairs and are therefore unsuitable for any other application. Furthermore, as they are single-sample instruments, measuring large numbers of samples is time-consuming. For laboratories using fluorescence for other applications, a microplate reader is a much better investment.

Fluorescence microplate readers offer a much higher throughput and more flexibility than compact tube fluorometers. The advantages of the microplate format and sample volume have already been discussed above. For fluorescence measurements, however, the sample volume is not so critical as the volume of reagent added to the sample must be taken into account to reach the minimum working volume of the microplate. DNA fluorescence quantification does not require path length correction because a standard curve is used to calculate concentrations instead of the Beer-Lambert formula.

Concerning the choice of microplate reader, most readers are suitable, if they are able to set the recommended excitation and emission wavelengths. Filter-based instruments offer higher sensitivity, while monochromator-based instruments provide more flexibility. Many common dyes for DNA quantification can be measured with the standard fluorescein filters, but before purchasing a DNA quantification kit, check if your instrument has suitable filters. If you are working close to the detection limit of the kit, you should be prepared to test other filter combinations to achieve the best signal-to-noise ratio.

Multimode Readers

Multimode microplate readers can be configured to measure both, absorbance and fluorescence, giving you the option to quantify DNA using either absorbance or fluorescence, as needed.

Comparison of instruments for DNA quantification

Instrument	Sample Format	Minimum sample volume	Advantages	Limitations
Microvolume spectrophotometer	Drop	1 µL (typical); 0.3 µL in some instruments	Quick and convenient Lowest sample volume	Low throughput Absorbance only
Microplate reader*	96-well plate	25-50 µL	High throughput Multimode readers can be used both for absorbance- and for fluorescence-based methods	Large sample volume may be needed depending on method and microplate Special microplates and pathlength correction may be needed for absorbance-based DNA quantification
	96-well plate, half area	15-25 µL
	384-well plate,	10-20 µL
	384-well plate, small volume	2-5 µL
	1536-well plate	1-2 µL
	Microvolume microplate	2 µL	Low sample volume Medium throughput	Handling might not be as convenient as with other methods
Tube fluorometer	0.5 mL tube	1-20 µL	Affordable Optimized for specific kits	Low throughput Fluorescence only Not suitable for other kits or applications

* Absorbance, fluorescence and multimode microplate readers are considered together

Featured products

Colibri+ LB 916 Microvolume Spectrophotometer

Tristar 5 Multimode Reader

μDrop Plate for low-volume measurements