Measuring the purity of low volumes of DNA at 4 °C using the Agilent Cary 60 UV-Vis spectrophotometer with fiber optics microprobe

Significance of the topic

The accurate measurement of DNA purity and concentration is a cornerstone of molecular biology, supporting applications from cloning and sequencing to quality control in pharmaceutical and biotech industries. UV-Vis spectrophotometry offers a rapid, non-destructive approach that minimizes sample consumption and consumable costs.

Objectives and study overview

This application note demonstrates a streamlined workflow for measuring low volumes (150 µL) of DNA at 4 °C directly in Eppendorf tubes, employing the Agilent Cary 60 UV-Vis spectrophotometer with a fiber optics microprobe. The goal is to validate accuracy, reproducibility, and time savings compared with traditional cuvette-based methods.

Methodology and instrumentation

DNA solutions of 0 – 50 µg/mL calf thymus DNA were prepared in Milli-Q water and kept at 4 °C. The Cary 60 system, equipped with a high-intensity xenon flash lamp, fiber optic probe and WinUV "RNA-DNA Estimation" software, recorded absorbance scans from 340 to 240 nm under normal laboratory lighting. Purity ratios (A260/A280) were automatically calculated with background subtraction at 320 nm. Blanks of Milli-Q water were measured between samples to correct for baseline.

Instrumentation used

• Agilent Cary 60 UV-Vis spectrophotometer with WinUV software and PC
• Fiber optic microprobe and probe coupler
• Quartz cuvettes (1 cm path length) for calibration

Main results and discussion

A linear calibration curve (Abs = 0.02004 × Conc) was obtained over 0 – 50 µg/mL with a correlation coefficient of 0.9976, confirming adherence to the Beer–Lambert law. Distinct absorbance peaks at 260 nm and accurate 260/280 purity ratios were recorded. The analysis of five samples required under two minutes, representing an estimated time saving of over 80% compared to conventional cuvette methods, while eliminating warming delays and reducing contamination risk.

Benefits and practical applications

• Eliminates cuvette handling and cleaning
• Direct measurement of cold samples at 4 °C
• Compatible with very small volumes (≥ 40 µL) or larger
• Non-destructive, cost-effective, and rapid workflow
• Adaptable for protein assays at 280 nm and for solids or gels via reflectance probes

Future trends and potential applications

Fiber-optic UV-Vis technology is poised for broader use in point-of-care testing, field deployments, and automated QC pipelines. Advances may include multiplexed detection, integration with robotic sample handlers, and real-time remote monitoring.

Conclusion

The Agilent Cary 60 UV-Vis spectrophotometer with fiber optics microprobe delivers fast, reliable, and reproducible DNA purity and concentration measurements at 4 °C directly in storage vessels, streamlining laboratory workflows and reducing operational costs.

References

• Warburg O. and Christian W. Biochem Z. 1942;310:384.
• Sambrook J., Fritsch EF., and Maniatis T. Molecular Cloning. 2nd ed.; Cold Spring Harbor Laboratory Press; 1989.
• Keighley B. and Fyfe D. Agilent Application Note #91; 1995.