Nucleic Acid Quantification
Convert a spectrophotometer A260 reading into DNA or RNA concentration using the standard absorbance factors (50 µg/mL for dsDNA, 40 µg/mL for RNA, 33 µg/mL for ssDNA). Enter the dilution factor used for the reading, and optionally an A280 to compute the A260/A280 purity ratio.
Calculator
Enter a reading and calculate to see the concentration
Concentration
Bench note
c (µg/mL) = A260 × dilution factor × factor
A 1.0 absorbance unit at 260 nm corresponds to 50 µg/mL double-stranded DNA, 33 µg/mL single-stranded DNA, or 40 µg/mL RNA. The dilution factor scales the reading back to the original sample. Yield = concentration × volume ÷ 1000.
About this calculation
What this is
Nucleic acids absorb at 260 nm, and the absorbance of a 1 mg/mL solution in a 1 cm path length is a published constant (50 for dsDNA, 40 for RNA, 33 for ssDNA). Measuring A260 in a diluted aliquot and multiplying by the dilution factor and the right constant yields the stock concentration.
Why it is used
Every downstream application — PCR, sequencing, transfection, in vitro transcription — needs an accurate nucleic acid concentration. A260 is the most accessible bench measurement, but the constant and dilution must be applied correctly.
Why this calculator exists
Mixing up the absorbance factor (50 vs 40 vs 33) or forgetting the dilution factor are the two classic bench errors. The calculator makes the constant explicit, multiplies by your dilution, and reports a purity ratio when you also provide an A280.
Key assumptions
- The spectrophotometer was blanked correctly with the same buffer the sample was diluted into.
- The sample is pure enough at A260 for Beer–Lambert to apply (A260 < ~1.5).
- The path length is 1 cm (or your instrument has been corrected for a different path length).
Limitations
- A260 cannot distinguish DNA from RNA — use a fluorometric assay for that.
- Contaminants such as phenol or carbohydrates inflate A260, producing overestimated concentrations.
- Very low concentrations (A260 < 0.05) are at the noise floor of standard spectrophotometers.
What this calculates
- Concentration µg/mL
- Analyte concentration in the original sample: A260 × dilution factor × conversion factor.
- Total yield µg
- Total mass of analyte in the final volume (shown when a volume is supplied).
- A260/A280 ratio
- Purity indicator (shown when A280 is supplied). Typical pure ranges: dsDNA 1.7–2.0, ssDNA 1.8–2.0, RNA 1.9–2.1.
Frequently asked questions
How do I convert an A260 reading into DNA or RNA concentration?
Multiply the blank-corrected A260 by the total dilution factor and by the conversion factor for your analyte: concentration (µg/mL) = A260 × dilution factor × factor. The calculator applies the right factor automatically once you pick the analyte, and reports the concentration of the original sample. Enter the final volume as well and it returns the total yield in µg.
What is the difference between the 50, 40, and 33 conversion factors?
These are the standard absorbance constants: a 1.0 A260 reading equals 50 µg/mL for double-stranded DNA, 40 µg/mL for RNA, and 33 µg/mL for single-stranded DNA. The factor you need depends only on the analyte you are measuring, so choose dsDNA, ssDNA, or RNA in the Analyte field. Applying the wrong constant is one of the two classic bench errors this calculator is designed to prevent.
What is a good A260/A280 ratio for pure nucleic acid?
The typical pure ranges are 1.7–2.0 for dsDNA, 1.8–2.0 for ssDNA, and 1.9–2.1 for RNA. Supply an optional A280 reading and the calculator reports the A260/A280 ratio and flags a result outside the range for your analyte, since protein or other contaminants can affect the reading. A low ratio is a purity warning rather than a broken sample, and the ratio cannot detect every contaminant.
Do I need to correct for the dilution I made before reading the sample?
Yes. Enter the total dilution applied to the sample before the reading, and the calculator multiplies it through so the reported concentration refers to your original tube rather than the diluted aliquot. Reading a 2 µL sample into 100 µL, for example, takes a dilution factor of 50. Forgetting that factor is the other classic bench error, and it under-reports concentration by exactly the dilution you applied.
What can A260 quantification not tell me about my sample?
It measures nucleic acid that absorbs near 260 nm, so contaminants such as protein, phenol, or free nucleotides inflate the apparent concentration, and the method cannot tell intact from degraded material. It also assumes a blank-corrected reading taken in a 1 cm path length, or equivalent microvolume optics. Use a gel or fragment-analysis run for integrity, and treat the purity ratio as a coarse screening tool.
For research and educational use only. Not for clinical or diagnostic decisions. Always verify calculations independently before use in critical applications.