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

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.

Fluorescence Readers

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

There are single tube fluorometers that use 0.5 mL PCR tubes as the sample vessel, and they are often offered by manufacturers of the DNA quantification kits. While they are very affordable, they can usually only measure at 2 wavelength pairs and are unsuitable for any other applications. They are also single-sample instruments and is time consuming when measuring large numbers of samples. 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. For fluorescence measurements the sample volume is not as critical as the volume of reagent added to the sample. This must be considered 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.

Most microplate 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 any DNA quantification kit check if your instrument has the correct filters. If you are working close to the detection limit of the kit, then be prepared to test other filter combinations to achieve the best signal-to-noise ratio.

Multimode Readers

Multimode microplate readers can give you the option to quantify DNA using either absorbance or fluorescence.

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 for both absorbance-and 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
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

μDrop Plate for low-volume measurements