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

Significance of the topic

The quantitation of nucleic acids in microliter volumes is critical for modern bioanalytical workflows, enabling higher throughput, cost efficiency and minimal sample consumption.

Aim and overview

This study demonstrates the capability of the Agilent Cary 50/60 UV-Vis spectrophotometer equipped with an ultra-microvolume cuvette to accurately and reproducibly measure DNA concentration in samples as small as 4 µL.

Used instrumentation

• Agilent Cary 50 UV-Vis spectrophotometer (compatible with Cary 60)
• Ultra-microvolume cuvette with 0.2–1.0 mm pathlength cap
• Xenon flash lamp light source with <1.5 nm spectral bandwidth slits
• WinUV software DNA/RNA module for absorbance ratio calculations

Methodology

DNA was purified from E. coli containing vector pBUDCE4.1 using an endotoxin-free plasmid kit. Four microliters of each sample were loaded onto the microvolume cuvette, aligned for >25% transmission at 500 nm, and scanned from 200 to 400 nm. Concentration was calculated from the 260/280 nm absorbance ratio, with background correction at 320 nm.

Main results and discussion

Repeated measurements of 4 µL undiluted DNA yielded an average concentration of 1224.5 µg/mL with 0.29% RSD, demonstrating high reproducibility. In comparison, 400 µL samples in a 1 cm cuvette produced 117.8 µg/mL with 0.17% RSD. The small beam geometry and optimized optics of the Cary 50/60 allowed reliable readings without sample dilution and extended photometric range above 150 Abs.

Benefits and practical applications

The approach offers rapid analyses (as little as two seconds per sample), accurate and non-destructive DNA quantitation with minimal consumables and cleaning. It supports high-throughput workflows in research, quality control and clinical laboratories.

Future trends and potential applications

Emerging directions include integration with microfluidic platforms, automated sample handling, expansion to protein and other biomolecule assays, and development of portable UV-Vis systems for point-of-care diagnostics.

Conclusion

The ultramicrovolume capability of Agilent Cary 50/60 UV-Vis instruments provides a robust, precise and efficient tool for DNA quantitation in volumes down to a few microliters.

References

1. Warburg O., Christian W. (1942) Biochem Z., 310, 384.