A260A280

A260/A280 is a widely used metric in molecular biology to assess the purity of nucleic acid samples, typically measured with a UV–visible spectrophotometer. The measurement compares absorbance at 260 nm, where nucleic acids absorb maximally, with absorbance at 280 nm, where proteins containing aromatic amino acids absorb light. The ratio is calculated by dividing A260 by A280.

Purity interpretation: For purified samples, the expected ratios are roughly 1.8 for double-stranded DNA and around 2.0 for RNA. Ratios significantly lower than these values can indicate contamination with proteins, phenol, or other UV-absorbing contaminants that absorb more at 280 nm; unusually high ratios might indicate contamination with substances that absorb at 260 nm or simply low protein content. The A260/A230 ratio is often evaluated alongside A260/A280 to assess contamination by organic compounds or salts; typical pure nucleic acids show A260/A230 around 2.0–2.2.

Concentration and measurement factors: The A260 reading is also used to estimate nucleic acid concentration using standard conversion factors (approximately 50 µg/mL per A260 unit for double-stranded DNA and about 40 µg/mL per A260 unit for RNA; values vary with preparation and instrument). The measurement should be performed on a clean, clear solution, with appropriate blanks and path lengths, typically 1 cm cuvettes or instrument-specific optics.

Limitations: The A260/A280 ratio is a useful quick check but does not guarantee suitability for all downstream applications. It can be affected by sample degradation, residual solvents, salts, or buffers; more sensitive methods such as fluorometric assays may be used for precise quantification and purity assessment.