Please be aware that this article was written some time ago. To ensure accuracy and relevance, please consult our experts to determine the most suitable spectrometer for performing these measurements currently.
Text was written on the base of Ocean Optics application note
DNA Measurements with the STS-UV Microspectrometer
Overview
DNA measurements typically involve samples at the extremes of the concentration range, with DNA suspended in solution at either very high or very low concentrations. While dilution is possible for concentrated DNA solutions, low concentration samples present more of a challenge. Without an adjustable pathlength spectrometer or a small volume sampler, the cuvette pathlength must be adjusted to measure a wide range of concentrations.
Advantages of the STS-UV Microspectrometer
The STS-UV microspectrometer, with its high dynamic range and signal-to-noise ratio, facilitates DNA measurements over a broad concentration range without requiring changes to the pathlength. This allows for absorbance levels ranging from 0.005 to over 1.5 absorbance units (AU).
Biological molecules are routinely measured using UV absorbance to obtain critical information on concentration and sample purity. With a wavelength range of 190 to 650 nm and approximately 1.5 nm optical resolution, the STS-UV microspectrometer is ideal for these measurements. It combines high performance in a compact form, providing high-quality UV absorbance data across a wide range of sample concentrations. This application note demonstrates its exceptional performance through absorbance measurements of DNA samples ranging from 0.15 to 150 μg/mL.
Techniques
Applications
- DNA quantification
- RNA quantification
- Protein quantification
- DNA purity
Measuring DNA Absorbance with the STS-UV Microspectrometerplication Note
Introduction:
Various concentrations of Salmon DNA (Sigma D-1626) were prepared in deionized water. DNA absorbance was measured in a 1 cm pathlength cuvette using an STS-UV and the deuterium lamp of a DH-2000-BAL balanced deuterium and tungsten halogen light source. Measurements were conducted with both the deuterium and tungsten halogen lamps and with just the deuterium lamp to demonstrate the impact of out-of-band light on the maximum absorbance level achieved with the spectrometer.
Results:
The absorbance spectra and absorbance at 260 nm (OD260) measured with the STS-UV for low concentrations of DNA (0.15 to 2.5 μg/mL DNA) are shown in Figures 1 and 2. Replicate measurements at these concentrations showed variability of less than 0.0004 AU (error bars fall within the data marker). The linearity of the low concentration data is demonstrated by a correlation coefficient value of 0.9999 for a linear least squares fit of these data points. As shown in Figure 1, the STS-UV provides repeatable, high-quality DNA absorbance data even with absorbance values as low as 0.005 AU.
Figure 1: Low concentration DNA solution absorbance measured with STS-UV
Figure 1: OD260 vs. DNA concentration in water ( from 0.15 to 2.5 μg/m)
The linearity and wide dynamic range of the STS-UV are further demonstrated in Figure 3 for DNA concentrations ranging from 0.15 to 110 μg/mL. The linearity of the data over this wide concentration and absorbance range is illustrated by an R² value of 0.9996. There is a slight flattening or roll-off in absorbance at 110 μg/mL, suggesting that absorbance is linear to somewhere between 100 and 110 μg/mL at approximately 1.6 AU.
Figure 1: OD260 vs. DNA concentration in water ( from 0.15 to 110 μg/m)
Figure 1: OD260 vs. DNA concentration in water ( from 0.15 to 150 μg/m)
The maximum absorbance measured with the STS-UV is shown in Figure 4. Absorbance values as high as approximately 2.1 AU were measured. As shown by the data points in red, the OD260 of DNA with concentration is very linear to approximately 1.6 AU between 100 and 110 μg/mL DNA. At higher absorbance values measured for DNA concentrations above 100 μg/mL, the flattening or roll-off in the graph suggests that the measurements are near the stray light limit for these measurements. Notably, this entire concentration range was measured using a 1 cm pathlength cuvette, without the need for pathlength adjustment to measure DNA absorbance at 260 nm ranging from 0.005 to 2.1 AU.
Conclusion:
With a dynamic range of 4600:1 and a signal-to-noise ratio of more than 1500:1, the STS-UV packs tremendous performance into a compact, powerful microspectrometer. Ideal for DNA, RNA, and protein determination, the STS-UV covers the entire range from 190 to 650 nm with approximately 1.5 nm optical resolution. As shown for these DNA samples ranging from 0.15 to 150 μg/mL, the STS-UV enables measurements over a wide absorbance range from 0.005 to 2.1 AU. The STS-UV simplifies UV absorbance measurements by enabling measurements over a wide concentration and absorbance range without the need for adjustable sampling accessories.
Note:
This article was written some time ago. To ensure accuracy and relevance, consult our experts to determine the most suitable spectrometer for performing these measurements currently.
