Simple and rapid quantitation of microliter DNA samples using the Agilent Cary 50/60 UV-Vis

Importance of Topic

In modern biotechnology and molecular biology, accurate quantitation of nucleic acids in very small volumes is critical to preserve precious samples, increase throughput and reduce costs. Traditional UV-Vis spectrophotometers require milliliter volumes, limiting applications in high-throughput genomics, diagnostic assay development and quality control in pharmaceutical research.

Study Goals and Overview

The primary objective was to evaluate the performance of the Agilent Cary 50/60 UV-Vis spectrophotometers equipped with an ultra-microvolume cuvette for DNA concentration measurements. The study focused on determining DNA sample purity and concentration via absorbance ratios at 260 nm and 280 nm, following the classic Warburg and Christian method.

Methodology and Instrumentation

• Instrumentation: Agilent Cary 50 (or Cary 60) UV-Vis system with high-intensity xenon flash lamp and <1.5 nm spectral bandwidth slits.
• Ultra-microvolume cuvette: 1.0 mm pathlength cap for sample volumes as low as 4 µL.
• Sample preparation: Plasmid DNA purified using Qiagen EndoFree Plasmid Giga Kit, eluted in endotoxin-free water; 4 µL loaded directly onto the cuvette surface.
• Measurement settings: Wavelengths at 260 nm and 280 nm; background correction at 320 nm; scan range 400–200 nm; fast scan rate; DNA/RNA module in Cary WinUV software.

Main Results and Discussion

Reproducibility was assessed via repeated scans with removal and replacement of the cuvette. For 4 µL DNA samples, the 260/280 ratio averaged 1.747 with a calculated concentration of 1224.5 ± 3.6 µg/mL (0.29% RSD). A comparison using 400 µL in a conventional 1 cm cuvette yielded 260/280 ratios around 1.63 and a concentration of 117.8 ± 0.2 µg/mL (0.17% RSD). The small beam geometry and optimized light throughput of the Cary 50/60 delivered high-quality spectra and minimal variability even at sub-10 µL volumes.

Benefits and Practical Applications

• Non-destructive, label-free quantitation requiring only a few microliters of sample.
• Fast analysis time—spectral scan and calculation in as little as two seconds.
• Extended photometric range (>150 Abs) enables direct measurement of undiluted or minimally diluted biological samples.
• Reduced consumable costs and cleaning requirements compared to quartz cuvettes.

Future Trends and Potential Applications

Ongoing developments may include further miniaturization of sample holders and integration with automated liquid handling and microfluidic platforms. The approach can be extended to protein quantitation and other biomolecules by adapting wavelength settings and software modules. Enhanced connectivity to laboratory information management systems (LIMS) and real-time data analytics may also streamline workflows in genomics, proteomics and clinical diagnostics.

Conclusion

This study demonstrates that the Agilent Cary 50/60 UV-Vis spectrophotometers, when paired with an ultra-microvolume cuvette, provide a robust, precise and rapid method for DNA quantitation in microliter volumes. The combination of high-intensity illumination and optimized optics achieves reproducibility better than 0.3% RSD, supporting high-throughput and low-volume applications without compromising data quality.

References

1. Warburg O, Christian W. Biochemische Zeitschrift. 1942;310:384.