Nucleic acid quantification on Lunatic using A260
Applications | 2020 | Unchained LabsInstrumentation
Accurate measurement of nucleic acid concentration and purity is essential in molecular biology workflows, impacting downstream applications such as PCR, sequencing and cloning. The use of UV/Vis absorbance at 260 nm (A260) provides a rapid and reliable approach to quantify dsDNA, ssDNA and RNA, ensuring reproducible results in research and quality control laboratories.
This application note demonstrates the implementation of classic A260 UV/Vis assays on the Lunatic and Little Lunatic systems. It outlines the procedure for sample selection, blank correction, data acquisition and purity assessment. A comparative case study with the NanoDrop™ 2000 further evaluates performance, linearity and accuracy.
Samples of dsDNA, ssDNA or RNA are pipetted onto the instrument slide and measured without additional user input, using sample buffer as blank. The baseline‐corrected absorbance at 260 nm is multiplied by a factor specific to the nucleic acid type (50 ng/µL per A260 for dsDNA, 33 for ssDNA, 40 for RNA). Turbidity correction via background subtraction improves accuracy. Purity ratios A260/A280 and A260/A230 are calculated to detect protein or chemical contaminants.
Both Lunatic systems display concentration values, spectra and purity ratios on-screen. Case study data show octuplicate dilution series for DNA and RNA against gravimetric standards. Lunatic and NanoDrop 2000 results align closely with target concentrations, exhibiting linear response (R2 ≈ 0.99) and minimal deviation. Purity ratios consistently fell within expected ranges (A260/A280 ~1.8–2.0; A260/A230 ~2.0–2.2).
Advances may include expanded wavelength assays for novel biomolecules, further miniaturization, integration with robotic sample handlers and cloud‐based data management. Enhanced algorithms for complex sample matrices and real‐time quality feedback could broaden the adoption in clinical diagnostics and high‐throughput screening.
The Lunatic and Little Lunatic platforms offer robust, user‐friendly solutions for nucleic acid quantification and purity analysis. Their performance matches that of established instruments, while delivering automation, versatility and comprehensive reporting in a streamlined workflow.
Unchained Labs. Application Note: Nucleic acid quantification on Lunatic using A260. Rev C, 2020.
UV–VIS spectrophotometry, Particle characterization
IndustriesProteomics
ManufacturerUnchained Labs
Summary
Significance of the Topic
Accurate measurement of nucleic acid concentration and purity is essential in molecular biology workflows, impacting downstream applications such as PCR, sequencing and cloning. The use of UV/Vis absorbance at 260 nm (A260) provides a rapid and reliable approach to quantify dsDNA, ssDNA and RNA, ensuring reproducible results in research and quality control laboratories.
Aims and Study Overview
This application note demonstrates the implementation of classic A260 UV/Vis assays on the Lunatic and Little Lunatic systems. It outlines the procedure for sample selection, blank correction, data acquisition and purity assessment. A comparative case study with the NanoDrop™ 2000 further evaluates performance, linearity and accuracy.
Used Instrumentation
- Lunatic UV/Vis spectrometer with classic A260 applications for high-throughput quantification
- Little Lunatic compact UV/Vis system with preconfigured A260 assays
- NanoDrop™ 2000 spectrophotometer for reference comparison
Methodology
Samples of dsDNA, ssDNA or RNA are pipetted onto the instrument slide and measured without additional user input, using sample buffer as blank. The baseline‐corrected absorbance at 260 nm is multiplied by a factor specific to the nucleic acid type (50 ng/µL per A260 for dsDNA, 33 for ssDNA, 40 for RNA). Turbidity correction via background subtraction improves accuracy. Purity ratios A260/A280 and A260/A230 are calculated to detect protein or chemical contaminants.
Main Results and Discussion
Both Lunatic systems display concentration values, spectra and purity ratios on-screen. Case study data show octuplicate dilution series for DNA and RNA against gravimetric standards. Lunatic and NanoDrop 2000 results align closely with target concentrations, exhibiting linear response (R2 ≈ 0.99) and minimal deviation. Purity ratios consistently fell within expected ranges (A260/A280 ~1.8–2.0; A260/A230 ~2.0–2.2).
Benefits and Practical Applications
- High-throughput, automated nucleic acid quantification requiring minimal user input
- Integrated purity assessment to flag contaminants early in workflows
- Flexible reporting options (HTML, XML, TXT, CSV, XLSX, PDF) for record‐keeping and data exchange
- Comparable accuracy and linearity to established microvolume spectrophotometers
Future Trends and Potential Uses
Advances may include expanded wavelength assays for novel biomolecules, further miniaturization, integration with robotic sample handlers and cloud‐based data management. Enhanced algorithms for complex sample matrices and real‐time quality feedback could broaden the adoption in clinical diagnostics and high‐throughput screening.
Conclusion
The Lunatic and Little Lunatic platforms offer robust, user‐friendly solutions for nucleic acid quantification and purity analysis. Their performance matches that of established instruments, while delivering automation, versatility and comprehensive reporting in a streamlined workflow.
Reference
Unchained Labs. Application Note: Nucleic acid quantification on Lunatic using A260. Rev C, 2020.
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