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Tutorials : Oct 1, 2012 ( )
Quantification of Microliter Volumes of DNA
An Alternative to Traditional Low Volume Spectroscopy Techniques Is Now Available
Direct measurement of DNA using UV/Vis spectroscopy remains one of the simplest ways to measure DNA concentration, and the ratio of absorptions at 260 nm vs. 280 nm is used to assess purity of DNA preparation. However, these techniques are not without problems. The high absorbance of DNA and proteins at such wavelengths means that traditional cuvettes are often unsuitable for measuring the high concentration levels without dilution of the sample. Not only is this time-consuming but it also leaves greater scope for error.
A new class of spectrophotometer has emerged to improve and streamline the measurement of such samples. These instruments negate the need for dilutions by measuring microvolume amounts of sample over very short pathlengths. They also reduce waste by using less sample, which is particularly beneficial when only a few microliters are available for measurement. However, difficulties in cleaning these devices and compromises to the accuracy of the optical pathlength do mean that dedicated low volume spectrophotometers frequently compromise ease of use and measurement accuracy.
BioDrop has developed an innovative, alternative approach to microvolume spectroscopy using a BioDrop microvolume cuvette for DNA measurement in a standard spectrophotometer such as the Biochrom Libra S60 duel beam instrument.
The BioDrop Cuvette consists of two precision engineered halves that are magnetically held together with a droplet of sample securely loaded in the middle (Figure 1). The Cuvette is composed of PEEK polymer which is more robust than conventional quartz cuvettes and other low-volume devices. The sample is measured through a large sample window made of quartz.
The BioDrop Cuvette 125, which has a 0.125 mm pathlength, is capable of holding samples as small as 0.6 μL, while the BioDrop Cuvette 500, with a 0.5 mm pathlength, holds samples as low as 2.5 μL. As the BioDrop Cuvette has the same dimensions as a standard cuvette, it is suitable for use in almost all spectrophotometers with a standard cuvette holder.
During use, light shines through the quartz window in the device and the optical pathlength of the measurement area is defined by a precisely machined spacer ring which is mounted on a thin membrane. This membrane provides enough pressure to overcome the surface tension of the sample and ensures that it fills the sample gap while any excess liquid is forced out thus the optical pathlength is accurate to within a few microns.
Measuring DNA Concentration
The DNA measured during this investigation was Sigma-Aldrich Item#1626-250MG; the BioDrop Cuvette 0.125 mm and the 0.5 mm pathlength devices were both measured using the same protocol. To obtain results, the appropriate BioDrop pathlength was automatically selected from a drop-down menu in onboard software of the Libra S60 dual beam spectrophotometer and five recorded measurements were averaged together.
Samples were then progressively diluted and the measurements repeated for all subsequent concentrations. The measured concentration was plotted against the expected value, along with the percent difference between the measured and expected values.
Repeatability was tested by measuring a single sample multiple times before calculating the peak and RMS variation between measurements. The BioDrop Cuvette was removed from the instrument between each run. The detection limit was also tested by performing a series of measurements on ultra-pure water and recording the reported concentrations.
When carrying out such investigations, contamination and cleanliness were of paramount importance, especially considering the small size of the samples being used. Care was taken during testing to ensure no bubbles or dust particles were caught in the pipetted volumes, while the BioDrop Cuvette’s large sample window allowed the user to see any physical contamination. Sample carry over was assessed by alternating between samples of ultra-pure water and concentrated DNA, wiping with a lint-free cloth after each measurement.
Figures 2 and 3 show the measured concentration against dilution factor. A linear least squares fit shows that both pathlength Cuvettes exhibit excellent linearity. A correlation of 0.9998 and 0.9997 was shown with the 0.5 mm and 0.125 mm pathlength BioDrop Cuvettes respectively, showing statistically accurate results with all measured concentrations.
A detection limit of 1.2 ng/µL was measured with the BioDrop Cuvette 500, while a detection limit of 7.1 ng/µL was measured with the BioDrop Cuvette 125. These were similar to the carry over testing results, indicating that contamination between successive samples is negligible when the user followed a simple cleaning protocol.
Finally, reproducibility tests showed a peak to peak value of 1.0 and a standard deviation of 0.5 for a 100 ng/µL concentration, followed by a peak to peak of 4.1 and a SD of 2.4 for a 1,000 ng/µL concentration when using the BioDrop Cuvette 500. When the same concentrations were measured with the BioDrop Cuvette 125, peak to peaks of 4.6 and 6.6 and standard deviations of 2.3 and 3.2 were measured for 100 ng/µL and 1,000 ng/µL respectively.
As well as demonstrating the consistency of results, this suggested that identical positioning of the cuvette does not significantly impact measurement results, as the BioDrop Cuvette was removed and replaced each time. There was no need for adapters with lenses or screw adjustments to ensure the light beam was correctly coupled with the device when it was reinserted.
The BioDrop Cuvette has proven itself able to overcome some of the most common disadvantages of conventional low volume spectroscopy techniques, providing a simple and effective way to quantify microliter volumes in almost any UV/Visible spectrophotometer. The tests of accuracy, precision and linearity of the BioDrop Cuvette 500 and the BioDrop Cuvette 125 confirmed high-quality measurement performance over a wide dynamic range when used with a Biochrom Libra S60 spectrophotometer.
Results were reliable, accurate, and repeatable, and experimental procedure was noticeably simplified in comparison to other common techniques. The BioDrop Cuvette appears to be ideally suited for low volume work in modern life science laboratories.
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