A260 Calculators

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A260 (absorbance at 260 nm) is the UV absorbance of a nucleic acid solution, used to quantify DNA and RNA based on the Beer-Lambert law. The aromatic rings of the nucleobases absorb strongly at 260 nm. Standard conversion factors: 1 A260 unit = 50 μg/mL dsDNA; 40 μg/mL for RNA; 33 μg/mL for ssDNA. Purity is assessed by the A260/A280 ratio (protein contamination) and A260/A230 ratio (chemical contamination). NanoDrop spectrophotometers are widely used for rapid, low-volume (1–2 μL) nucleic acid quantification in the 2–3000 ng/μL range.

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A260 Quantification Formulas

  • dsDNA: concentration (μg/mL) = A260 × 50 × dilution factor
  • ssRNA: concentration (μg/mL) = A260 × 40 × dilution factor
  • ssDNA: concentration (μg/mL) = A260 × 33 × dilution factor
  • Oligonucleotides: use extinction coefficient calculated from sequence composition

Example: A260 = 0.35 for undiluted dsDNA sample: concentration = 0.35 × 50 = 17.5 μg/mL = 17.5 ng/μL.

Purity Ratios

A260/A280 ratio:

  • Pure dsDNA: ~1.8
  • Pure RNA: ~2.0
  • Below target: protein or phenol contamination
  • Above 2.0 (in DNA): possible RNA contamination

A260/A230 ratio:

  • Pure nucleic acid: 2.0–2.2
  • Below 1.8: guanidinium salts, EDTA, phenol, carbohydrates — inhibits downstream applications

NanoDrop Instrument

NanoDrop measures absorbance of a 1 μL sample in a 1 mm path (using surface tension). Automatically applies conversion factors and calculates purity ratios. Can accurately measure 2–3700 ng/μL for dsDNA. Path length variation is the main source of error for very dilute samples (<5 ng/μL). For concentrations below NanoDrop detection limit or when specificity for intact dsDNA is needed: use Qubit fluorometric assay (PicoGreen for dsDNA; RiboGreen for RNA).

Limitations

A260 cannot distinguish DNA from RNA (both absorb at 260 nm). Degraded nucleotides also absorb at 260 nm — A260 measurement overestimates intact nucleic acid concentration if degradation has occurred. Free nucleotides (from enzymatic reactions) inflate A260 readings.

Glossary

A260
Absorbance at 260 nm; used to quantify nucleic acids: 1 A260 unit = 50 μg/mL dsDNA, 40 μg/mL RNA, 33 μg/mL ssDNA; linear range 0.1–0.8 (NanoDrop auto-adjusts path length).
A260/A280 Ratio
Nucleic acid purity metric: pure dsDNA ≈ 1.8; pure RNA ≈ 2.0; significantly lower values indicate protein or phenol contamination; measured simultaneously with A260.
Qubit Fluorometric Assay
Uses dye-specific fluorescence (PicoGreen for dsDNA, RiboGreen for RNA) for accurate quantification; distinguishes DNA from RNA; better for low concentrations and mixed samples than A260.

Frequently Asked Questions

Concentration (μg/mL) = A260 × conversion factor × dilution factor. Conversion factors: dsDNA = 50; ssRNA = 40; ssDNA = 33. Example: A260 = 0.42 for a sample diluted 10-fold; dsDNA: concentration = 0.42 × 50 × 10 = 210 μg/mL. On NanoDrop: select nucleic acid type before measurement — the software applies the correct conversion factor automatically. Always ensure A260 is in the linear range (0.1–0.8 on standard 1 cm path; NanoDrop adjusts path length automatically).

A260/A280: protein contamination ratio. Target: dsDNA ≈ 1.8; RNA ≈ 2.0. Proteins absorb at 280 nm (Trp, Tyr). Ratio < target: protein or phenol contamination → may inhibit PCR, restriction enzymes, transfection. A260/A230: detects co-purification of guanidinium salts, EDTA, phenol, or carbohydrates (all absorb around 230 nm). Target: 2.0–2.2. Ratio < 1.8: inhibitory contaminants present — may affect downstream applications. Low A260/A230 is common with column-based RNA extractions if wash steps are insufficient or elution volume is too small.

Qubit (fluorometric quantification) is preferred when: (1) Samples are mixed (contain both DNA and RNA) — Qubit uses dye-specific for dsDNA (PicoGreen) or RNA (RiboGreen), distinguishing them; NanoDrop cannot. (2) Concentration is very low (<5 ng/μL) — NanoDrop is inaccurate below this threshold due to path length variation. (3) Samples contain degraded nucleic acids — Qubit dye binds to intact double-stranded molecules, giving a more accurate estimate of functional template. (4) Downstream applications require accurate dsDNA (e.g., NGS library preparation where over- or under-loading of the sequencer is costly). NanoDrop is faster and simpler for routine quality checks on clean samples.

RNA uses 40 μg/mL per A260 unit vs. 50 for dsDNA because: (1) Single-stranded RNA lacks the stacking interactions of double-stranded DNA — nucleobases in ssDNA and ssRNA are more exposed to solvent, changing their extinction coefficient. (2) The uracil base in RNA has slightly different UV absorption properties than thymine in DNA. (3) Average molar extinction coefficients per nucleotide differ: RNA nucleotides have slightly lower average ε than DNA nucleotides on a mass basis. These are empirical conversion factors validated across many nucleic acid preparations — they are approximations that work well for typical samples but may deviate for sequences with unusual base compositions.