A260/A280 Ratio Calculators

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The A260/A280 ratio is the most commonly used quick indicator of nucleic acid purity in molecular biology. It compares UV absorbance at 260 nm (where nucleic acids absorb strongly) to absorbance at 280 nm (where proteins absorb, due to tryptophan and tyrosine). A ratio near 1.8 indicates pure DNA; near 2.0 indicates pure RNA. Values significantly below these suggest contamination with protein, phenol, or other UV-absorbing substances that can inhibit PCR, sequencing, and other downstream applications.

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What Is the A260/A280 Ratio?

UV absorbance measurements at two key wavelengths reveal sample purity:

  • A260: Nucleic acids absorb strongly at 260 nm due to aromatic base ring structures (purines and pyrimidines)
  • A280: Proteins absorb at 280 nm primarily due to tryptophan (~280 nm) and tyrosine (~274 nm) residues

A high ratio = more nucleic acid relative to protein = purer sample.

Expected A260/A280 Values

  • Pure double-stranded DNA: ~1.8
  • Pure RNA: ~2.0–2.1
  • Pure protein: ~0.5–0.8

Values are pH-dependent — measurements in slightly alkaline buffer (10 mM Tris, pH 8.0) give the most reliable results. Acidic pH depresses the ratio artificially.

Causes of Low A260/A280

  • Protein contamination: Increases A280 relative to A260. Re-purify with silica column or additional phenol-chloroform extraction.
  • Phenol carryover: Residual phenol absorbs at ~270 nm. Severely inhibits PCR at trace concentrations. Indicated by both low A260/A280 and low A260/A230.
  • Ethanol carryover: From precipitation or column washing. Primarily inhibits enzymes rather than affecting ratios significantly.

The A260/A230 Ratio

The A260/A230 ratio (expected 2.0–2.2) detects contamination with substances absorbing at 230 nm: guanidinium salts (from lysis buffers), EDTA, carbohydrates, and phenol. A low ratio (<1.8) indicates these inhibitors remain, often from incomplete silica column washing.

NanoDrop Limitations

NanoDrop instruments require only 1–2 μL and report concentration and purity ratios automatically. Key limitation: NanoDrop measures total A260 absorbance — it cannot distinguish between intact DNA, RNA, free nucleotides, or other UV-absorbing contaminants. For critical applications (NGS library prep), use fluorometric quantification (Qubit) for DNA-specific, accurate concentration measurement.

Glossary

A260/A280 Ratio
Ratio of UV absorbance at 260 nm (nucleic acids) to 280 nm (proteins). Pure dsDNA ~1.8; pure RNA ~2.0. Values below these indicate protein or phenol contamination that may inhibit downstream reactions.
A260/A230 Ratio
Ratio of absorbance at 260 nm to 230 nm. Expected 2.0–2.2 for pure nucleic acids. Low values indicate guanidinium salts, EDTA, phenol, or detergent contamination from extraction buffers.
NanoDrop
A micro-volume spectrophotometer measuring UV-Vis absorbance in 1–2 μL samples. Reports nucleic acid concentration and A260/A280 and A260/A230 ratios. Cannot distinguish between different UV-absorbing molecules — fluorometric quantification (Qubit) is more accurate for specific DNA/RNA measurement.

Frequently Asked Questions

Pure double-stranded DNA has an A260/A280 ratio of approximately 1.8 when measured in a slightly alkaline buffer (pH 7.5–8.0). Values significantly below 1.8 (e.g., 1.5–1.6) indicate protein or phenol contamination increasing A280. Values above 2.0 may suggest RNA contamination. Pure RNA has an expected ratio of ~2.0–2.1.

Common causes: protein contamination (increases A280); residual phenol from extraction (absorbs at ~270 nm, depresses ratio); and low DNA concentration (ratio becomes unreliable below A260 ~0.1). Re-purify using a silica column cleanup kit or additional phenol-chloroform extraction. Low pH also artificially depresses the ratio — always measure in Tris buffer at pH 7.5–8.0.

The A260/A230 ratio (expected 2.0–2.2) detects contamination with substances absorbing at 230 nm — guanidinium salts (from column lysis buffers), EDTA, carbohydrates, and phenol. A ratio below ~1.8 indicates these inhibitors are present in the sample. They can severely inhibit PCR, reverse transcription, and restriction enzyme digestion even when the A260/A280 looks acceptable.

Use NanoDrop for quick purity assessment (A260/A280 and A260/A230 ratios) and rough concentration estimates. Use Qubit (or another fluorometric assay) when accurate DNA-specific concentration is critical — for example, before NGS library preparation. NanoDrop measures all UV-absorbing material at 260 nm and overestimates concentration when RNA, free nucleotides, or other contaminants are present.