Nucleic Acid Concentration from A260 Calculator
Nucleic Acid Concentration from A260 Calculator
Turn an A260 reading into a DNA or RNA concentration with the appropriate extinction factor, and read the A260/A280 ratio alongside it — absorbance tells you how much is there, not whether it is intact.
Nucleic Acid Concentration from A260
A260, factor, dilution → µg/mLA260 0.42, double-stranded DNA, diluted 1:20 before reading, A280 0.23
Formula
factor = 50 for dsDNA · 33 for ssDNA · 40 for RNA
µg/mL and ng/µL are numerically identical
- A260
- absorbance at 260 nm, the peak of the purine and pyrimidine rings, in a 1 cm path length. Keep the reading below about 1.0: beyond that the relationship with concentration is no longer linear on most instruments and the result is an underestimate
- factor
- the concentration giving an absorbance of 1.0. Double-stranded DNA takes 50 µg/mL, single-stranded DNA 33 and RNA 40, because base stacking in a duplex suppresses absorbance — the hypochromic effect — so the same mass of single-stranded material absorbs more
- dilution factor
- the number of times the original sample was diluted before reading. This is the commonest source of error on the page: a tenfold slip here moves the answer by an order of magnitude and looks entirely plausible
- A260/A280
- the purity ratio for protein and phenol. About 1.8 is expected for DNA and about 2.0 for RNA; a low ratio indicates protein or phenol carryover. It is insensitive — substantial contamination is needed before the ratio moves much
- A260/A230
- the second purity ratio, expected at about 2.0–2.2. A low value points to guanidine salts, phenol or carbohydrate carried over from the extraction, all of which inhibit PCR and reverse transcription even when the A260/A280 ratio looks perfect
Worked example
A260 0.42, double-stranded DNA, diluted 1:20 before reading, A280 0.23
Concentration = 0.42 × 50 × 20 = 420 µg/mL, which is the same thing as 420 ng/µL
A260/A280 = 0.42 ÷ 0.23 = 1.83, within the 1.8-ish window expected for DNA
If the same readings had come from an RNA preparation, the factor would be 40 and the concentration 0.42 × 40 × 20 = 336 µg/mL — and the 1.83 ratio would now be low, because RNA is expected at about 2.0
For single-stranded DNA the factor is 33, giving 0.42 × 33 × 20 = 277.2 µg/mL
Note what none of this establishes: a completely degraded sample gives the same absorbance as an intact one of the same mass, and a DNA preparation heavily contaminated with RNA reads as a more concentrated DNA preparation
Extinction factors and what they assume
| Species | µg/mL per A260 unit | Why it differs |
|---|---|---|
| Double-stranded DNA | 50 | Base stacking in the duplex suppresses absorbance, so more mass is needed for the same reading |
| Single-stranded DNA | 33 | No duplex hypochromicity, so the same mass absorbs about 1.5 times as strongly |
| RNA | 40 | Single-stranded but with extensive local secondary structure, giving an intermediate figure |
| Oligonucleotide | Use the sequence-specific ε | The generic 33 is a poor approximation for a short oligo; base composition dominates at that length and suppliers quote a calculated extinction coefficient |
Reading the two purity ratios
| Ratio | Expected | A low value means | A high value means |
|---|---|---|---|
| A260/A280 | ≈ 1.8 for DNA, ≈ 2.0 for RNA | Protein or phenol carryover | For DNA, usually RNA carryover; otherwise a blanking artefact |
| A260/A230 | ≈ 2.0–2.2 | Guanidine salts, phenol or carbohydrate from the extraction — all PCR inhibitors | Usually a blank or baseline problem rather than anything real |
What absorbance can and cannot tell you
Nucleic acids absorb ultraviolet light with a peak at 260 nm, and over the linear range of a spectrophotometer that absorbance is proportional to concentration. The constant of proportionality is conventionally expressed the other way round, as the concentration giving an absorbance of 1.0 in a 1 cm path: 50 µg/mL for double-stranded DNA, 33 for single-stranded DNA, 40 for RNA. Multiply the reading by that factor and by the dilution used, and you have the concentration of the original sample. Because a microgram per millilitre is the same as a nanogram per microlitre, the number can be carried straight into a reaction set-up without conversion.
The two purity ratios come free with the measurement. A260/A280 is expected at about 1.8 for DNA and about 2.0 for RNA; a value below that indicates protein or residual phenol, both of which absorb strongly at 280 nm. A260/A230 is expected at roughly 2.0 to 2.2, and a low value points to guanidine salts, phenol or carbohydrate carried through from the extraction. The second ratio deserves more attention than it usually gets, because the contaminants it detects are precisely those that inhibit polymerases and reverse transcriptases, and it falls before A260/A280 does.
The limitations matter more than the arithmetic. Absorbance cannot distinguish intact nucleic acid from degraded nucleic acid: a sample sheared into fragments absorbs exactly as much as the intact material it came from, so a high concentration says nothing about whether the sample will support long-range amplification or library preparation. Absorbance also cannot distinguish DNA from RNA. Both absorb at 260 nm, so a DNA preparation contaminated with RNA reads as a more concentrated DNA preparation, and the only hint is an A260/A280 ratio that has drifted upward.
Where those distinctions matter, absorbance is the wrong measurement. A fluorometric assay using a dye that binds only double-stranded DNA, or only RNA, reports the species you asked about and ignores the other — which is why a fluorometric figure is often substantially lower than the A260 figure from the same tube, and why library preparation protocols specify one. For integrity, run a gel or an automated electrophoresis trace and look at the distribution of fragment sizes. Absorbance answers one question, how much ultraviolet-absorbing material is in the tube, and answers it quickly and cheaply.
Frequently asked questions
How do you calculate DNA concentration from A260?
Multiply the A260 reading by 50 µg/mL for double-stranded DNA and by the dilution factor used before reading. An A260 of 0.42 on a 1:20 dilution gives 0.42 × 50 × 20 = 420 µg/mL, which is the same as 420 ng/µL. Use 33 for single-stranded DNA and 40 for RNA.
What should the A260/A280 ratio be?
About 1.8 for DNA and about 2.0 for RNA. A lower ratio indicates protein or phenol carryover. The ratio is insensitive, so a value in the expected window does not prove the sample is clean — check A260/A230 as well, which should be roughly 2.0 to 2.2.
What does a low A260/A230 ratio mean?
Carryover of guanidine salts, phenol or carbohydrate from the extraction. These are potent inhibitors of polymerases and reverse transcriptases, so a low A260/A230 often predicts a failed or delayed amplification even when the A260/A280 ratio looks perfectly normal.
Can absorbance tell whether my DNA is degraded?
No. Degraded and intact nucleic acid of the same mass absorb identically at 260 nm, because the bases are unchanged. Integrity has to be assessed by gel electrophoresis or an automated fragment-size trace, not by a spectrophotometer reading.
Why does a fluorometric assay give a lower concentration than A260?
Because a fluorometric dye binds only the species it is designed for, usually double-stranded DNA, while A260 counts every ultraviolet-absorbing molecule in the tube — RNA, free nucleotides and single-stranded material included. The fluorometric figure is generally the one to use for library preparation.
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References
- Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press; 2001 — appendix on spectrophotometric quantification.
- Wilfinger WW, Mackey K, Chomczynski P. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques. 1997;22(3):474–481.
- Bustin SA, Benes V, Garson JA, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55(4):611–622 — on template quality and quantification.
Medical Disclaimer: The tools and content provided here are for educational and reference purposes only. They are not intended to substitute for professional medical advice, diagnosis, or treatment. Clinical decisions should always be based on the comprehensive assessment of a qualified healthcare professional.