NanoDrop Spectrophotometry

Main Findings:

• NanoDrop spectrophotometer uses surface tension to hold 1-2 µL samples without cuvette
• Optical path length automatically determined based on sample volume
• Capable of measuring nucleic acids from 2 ng/µL to 15,000 ng/µL
• 260/280 and 260/230 ratios calculated automatically for purity assessment
• Measurement takes <5 seconds per sample with high reproducibility

Relevance to Course: Demonstrates the fundamental operating principles of NanoDrop technology, essential for students to understand the measurement technique they're using.

Main Findings:

• Validation using NIST SRM 2372 DNA proved the method is safe and reliable
• Recovery percentage for the NIST SRM 2372 DNA was between 98% and 102% (acceptance criteria % Rec = 100% ± 5%)
• Recovery for Sprague Dawley rats and human DNA between 85% and 115% (acceptance criteria % Rec = 100% ± 15%)
• Method demonstrates excellent accuracy and precision for DNA quantification
• Linearity confirmed across concentration range 2-5000 ng/µL

Relevance to Course: Provides validation data demonstrating that NanoDrop measurements are accurate and reproducible, building student confidence in their results.

Main Findings:

• No significant difference between DeNovix and NanoDrop in estimated gDNA concentrations
• Spectrophotometry methods estimated close to or equal to 2 times higher concentrations compared to Qubit for pure DNA
• DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77)
• A260/280 and A260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions
• NanoDrop and DeNovix are interchangeable for routine DNA quantification

Relevance to Course: Demonstrates that NanoDrop provides accurate concentration measurements and that spectrophotometric methods may overestimate concentration compared to fluorometric methods, important for students to understand when comparing results.

Main Findings:

• Leaching of compounds from plastic tubes during storage can lead to overestimation of DNA concentrations up to 55% for polypropylene tubes
• Some plastic tubes can cause overestimation up to 300%
• Effect increases with storage time (days to weeks)
• Use of glass tubes or low-binding plastic recommended for long-term storage
• Fresh extracts provide most accurate measurements

Relevance to Course: Important for students to understand that storage conditions can affect measurement accuracy, emphasizing importance of measuring DNA soon after extraction.

Main Findings:

Spectrophotometric Methods (NanoDrop):

• Measure all nucleic acids (DNA + RNA)
• Sensitive to contaminants that absorb at 260 nm
• Typically overestimate concentration by 1.5-2x compared to fluorometry
• Best for purity assessment (260/280, 260/230 ratios)

Fluorometric Methods (Qubit):

• Measure only dsDNA (with dsDNA-specific dyes)
• Less affected by contaminants
• More accurate for PCR/sequencing applications
• Cannot assess purity ratios

Recommendation:

• Use both methods complementarily
• NanoDrop for purity assessment
• Qubit for accurate concentration for PCR setup

Relevance to Course: Teaches students that different quantification methods have different strengths and that combining methods provides most complete quality assessment.

Based on the literature reviewed, NanoDrop spectrophotometry offers:

Advantages:

• Microvolume measurement (1-2 µL)
• No cuvettes or consumables required
• Rapid measurement (5 seconds per sample)
• Wide dynamic range (2-15,000 ng/µL)
• Simultaneous purity ratio calculation
• High sample throughput

Limitations:

• Cannot distinguish DNA from RNA without RNase treatment
• Sensitive to contaminants that absorb at 260 nm
• May overestimate concentration in presence of RNA or contaminants
• Requires clean measurement surfaces
• Less accurate than fluorometry for low concentrations (<10 ng/µL)

Best Practices:

• Clean measurement pedestals between samples
• Use low-volume setting (1-2 µL) for precious samples
• Blank with elution buffer, not water
• Allow samples to equilibrate to room temperature
• Repeat measurements for critical samples (coefficient of variation should be <5%)

NanoDrop Measurement Principles:

1. Sample Loading: 1-2 µL placed on lower pedestal
2. Column Formation: Surface tension forms liquid column when upper pedestal lowered
3. Light Path: UV-Vis light passes through liquid column
4. Detection: Spectrometer measures absorbance at multiple wavelengths
5. Analysis: Software calculates concentration and purity ratios

Key Wavelengths:

• 260 nm: Maximum absorbance for nucleic acids
• 280 nm: Absorbance for proteins and phenol
• 230 nm: Absorbance for organic contaminants (chaotropic salts, carbohydrates)
• 320 nm: Background turbidity

Quality Metrics Interpretation:

260/280 Ratio:

• 1.8: Pure DNA
• 1.6-2.0: Acceptable
• <1.6: Protein or phenol contamination
• >2.0: RNA contamination or high pH

260/230 Ratio:

• 2.0-2.2: Pure DNA
• 1.8-2.0: Acceptable
• <1.8: Chaotropic salt or organic contamination

260/320 Ratio:

• Used to detect turbidity from cellular debris
• Should be >2.0 for clear samples
• Lower values indicate particulate contamination

When to Use Each Method:

Use NanoDrop for:

• Initial quality assessment of DNA extractions
• Purity ratio determination
• High-concentration samples (>10 ng/µL)
• Rapid screening of multiple samples
• When sample volume is very limited

Use Qubit for:

• Accurate quantification for PCR/qPCR setup
• NGS library quantification
• Low-concentration samples (<10 ng/µL)
• When precision is critical
• Distinguishing DNA from RNA

Use Both for:

• Comprehensive quality assessment
• Critical applications (NGS, cloning)
• Troubleshooting extraction protocols
• Publication-quality data

All citations verified and corrected on: November 5, 2025

Verification method:

• DOIs checked and confirmed to resolve to correct papers using reference verification tools
• Journal names and years cross-referenced via PubMed and CrossRef
• Main findings summarized from peer-reviewed sources
• Technical specifications verified against manufacturer documentation

High-confidence citations:

• Desjardins & Conklin (2010) - JoVE DOI 10.3791/2565 verified (foundational paper)
• García-Alegría et al. (2020) - International Journal of Analytical Chemistry DOI 10.1155/2020/8896738 verified
• Aranda et al. (2024) - PLOS ONE DOI 10.1371/journal.pone.0305650 verified (recent validation)
• Technical resources from Thermo Fisher verified 2024

Corrections made:

• Updated citation #2: Changed from Koetsier & Cantor (2019) to García-Alegría et al. (2020)
• Corrected DOI from 10.1155/2019/2304621 to 10.1155/2020/8896738
• Corrected article ID from 2304621 to 8896738
• Verified all DOIs against PubMed/CrossRef databases

Recommendation: These citations and technical information are suitable for student handouts and represent current best practices for NanoDrop spectrophotometry. Students should be encouraged to understand both the advantages and limitations of the technique.