Calculate DNA and RNA concentration from A260 absorbance readings at 260 nm using standard conversion factors.
Last updated: March 2026
DNA concentration measurement using UV spectrophotometry is the standard method for quantifying nucleic acids in molecular biology. This technique exploits the fact that nucleic acids absorb ultraviolet light maximally at 260 nanometers (nm) due to the aromatic rings in purine and pyrimidine bases.
The Beer-Lambert Law states that absorbance is directly proportional to concentration. Each type of nucleic acid has a specific extinction coefficient: double-stranded DNA (dsDNA) at 50 µg/mL, single-stranded DNA (ssDNA) at 33 µg/mL, and RNA at 40 µg/mL. These values mean that a solution with an A260 of 1.0 contains that concentration of nucleic acid.
The A260/A280 ratio provides critical information about sample purity. Pure DNA has a ratio of ~1.8, while pure RNA has a ratio of ~2.0. Deviations indicate contamination: ratios below 1.7 suggest protein contamination (proteins absorb at 280nm), while ratios above 2.0 may indicate RNA contamination in DNA samples.
Calculate the concentration of a dsDNA sample:
UV spectrophotometry estimates nucleic acid concentration using the Beer-Lambert Law. The law states that absorbance increases in direct proportion to the concentration of an absorbing substance and the distance light travels through the sample.
If the extinction coefficient is 1.2 L·mol⁻¹·cm⁻¹, the concentration is 0.8 mol/L, and the path length is 1 cm:
DNA and RNA have different average extinction properties because their structures and base arrangements affect how strongly they absorb ultraviolet light. For routine measurements normalized to a 1 cm optical path length, an A260 reading of 1.0 corresponds approximately to:
These are standard approximate conversion factors for routine nucleic acid quantification. Exact absorbance can vary with sequence composition, pH, contaminants, instrument settings, and sample condition.
The best measurement method depends on whether you need a rapid estimate of total nucleic acid, highly specific quantification, or information about DNA integrity.
| Method | Best For | Main Limitation |
|---|---|---|
| UV spectrophotometry Example: NanoDrop | Rapid measurement of total nucleic acid concentration and purity ratios | Other nucleic acids and UV-absorbing contaminants can increase the reading |
| Fluorometry Example: Qubit | Sensitive and selective DNA or RNA quantification, especially at low concentrations | Requires assay-specific fluorescent dyes, standards, and incubation |
| Agarose gel electrophoresis | Evaluating DNA size, degradation, integrity, and approximate quantity | Less precise and not primarily designed for concentration measurement |
Use UV spectrophotometry when you need a fast estimate of concentration and purity. Use fluorometry when accurate, nucleic-acid-specific quantification is important, particularly for low-concentration samples or sequencing workflows. Use gel electrophoresis when you also need to assess fragment size or degradation.
The A260/A280 ratio mainly helps identify protein contamination, while the A260/A230 ratio can reveal contamination from chemicals commonly carried over during nucleic acid extraction.
| A260/A230 Ratio | Interpretation |
|---|---|
| Approximately 2.0–2.2 | Generally consistent with a relatively pure nucleic acid sample |
| Lower than expected | May indicate phenol, guanidine salts, carbohydrates, EDTA, residual extraction reagents, or other compounds absorbing near 230 nm |
Purity ratios are screening indicators rather than definitive contamination tests. Very dilute samples can produce unstable ratios, and buffer composition, pH, blanking errors, and pedestal cleanliness can affect the result. Consider the complete absorbance spectrum and the requirements of the downstream application.
Nucleic acids absorb maximally at 260nm due to aromatic bases, while proteins absorb at 280nm due to aromatic amino acids (tryptophan, tyrosine). The 260/280 ratio reveals whether your sample contains protein contamination.
The optimal absorbance range is 0.1-1.0 for accurate measurements. If your reading is outside this range, dilute (if too high) or concentrate (if too low) your sample and remeasure, then apply the dilution factor to your calculation.
The conversion factors reflect differences in extinction coefficients. DNA's double helix has stacked bases that absorb less efficiently (factor 50) than single-stranded structures. ssDNA (33) and RNA (40) have more exposed bases and different base compositions affecting absorption.
A low ratio (<1.7) suggests protein contamination - try phenol-chloroform extraction or column purification. A high ratio (>2.0 for DNA) may indicate RNA contamination (use RNase treatment) or phenol carryover (re-extract or precipitate).
Multiply the A260 reading by 50 (for dsDNA) and by the dilution factor: concentration = A260 × 50 × dilution factor.
Yes, UV spectrophotometry is quantitative and follows the Beer-Lambert Law. However, it measures total nucleic acid and cannot distinguish between intact and degraded DNA. For quality assessment, combine with gel electrophoresis or fluorometry.
Nanograms per microliter (ng/µL) is numerically equivalent to micrograms per milliliter (µg/mL). These are the standard units for DNA concentration. To convert to molarity, you need to know the molecular weight (length) of your DNA.
The A260/A280 ratio provides a qualitative assessment. For precise quantification of contaminants, you'd need additional measurements (e.g., A230 for salts/chaotropes) or alternative methods like fluorometry (Qubit) which is DNA-specific and ignores contaminants.
Blanking with your dilution buffer (water, TE, etc.) establishes a baseline absorbance of zero for the buffer alone. This ensures you're only measuring absorbance from the nucleic acids, not from the buffer or the cuvette itself.
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