DNA and RNA A260 Concentration and Yield Calculator
Convert blank-corrected 260 nm ultraviolet absorbance readings into nucleic acid mass concentration and total eluted yield using standard conversion factors and an explicit optical path length. This reference explains microvolume 1 cm path-length normalisation and why A260/A280 and A260/A230 ratios are diagnostic observations rather than purity guarantees.
Opens the A260 card of the absorbance calculator with an illustrative reading of 0.75 at a 1 cm path for double-stranded DNA, which the rule of thumb converts to 37.5 µg/mL. The factors are 50 µg/mL for dsDNA and 40 for RNA from a Thermo Fisher technical page; the 33 for ssDNA is the convention NanoDrop instruments apply (Thermo Fisher, read 17 October 2026).
Check path-length conventions on microvolume and cuvette instruments
In a standard 1.0 cm quartz cuvette at neutral pH, an absorbance of 1.0 at 260 nm corresponds to approximately 50 µg/mL (50 ng/µL) of double-stranded DNA, 40 ng/µL of single-stranded RNA, or 33 ng/µL of single-stranded DNA (the ssDNA factor is the NanoDrop convention; Cavaluzzi and Borer measured 38 for non-repetitive ssDNA, and oligonucleotides need their own extinction coefficient). Microvolume pedestal spectrophotometers measure across a short liquid column of 1.0 mm (0.1 cm) or less to avoid diluting concentrated preps, but their export software usually multiplies the raw absorbance by ten and displays the 1.0 cm equivalent value.
Always verify whether your exported A260 column is raw or 1 cm normalised before entering the path length. Pairing a 1 cm normalised reading with a 0.1 cm path setting multiplies the concentration by an erroneous factor of ten.
Interpret A260/A280 and A260/A230 ratios alongside blank identity
Pure dsDNA typically exhibits an A260/A280 ratio near 1.85 and pure RNA near 2.0 in weakly alkaline buffer, while A260/A230 ratios below 2.0 often reflect carryover of guanidinium thiocyanate, EDTA, carbohydrates or phenol from extraction columns. Always blank the instrument with the exact elution buffer rather than water when samples are eluted in Tris or TE.
Equations and variables
Nucleic acid mass concentration from A260
cmass (µg/mL) = (A260 × d × CF) / l
- cmass
- Estimated nucleic acid mass concentration (1 µg/mL equals 1 ng/µL) (µg/mL)
- A260
- Blank-corrected absorbance at 260 nm at the stated path length l
- d
- Sample dilution factor applied prior to measurement
- CF
- Rule-of-thumb factor (50 for dsDNA, 40 for RNA, 33 for ssDNA, the NanoDrop convention) (µg mL⁻¹ cm)
- l
- Effective optical path length of the reading (cm)
Total eluted nucleic acid yield
myield (µg) = cmass (ng/µL) × Velution (µL) / 1000
- myield
- Total recovered nucleic acid mass in the elution tube (µg)
- cmass
- Concentration expressed in nanograms per microlitre (ng/µL)
- Velution
- Total recovered volume of eluate (µL)
Scope and limitations
- The conversion factors (50, 40 and 33 µg/mL per A260 unit) are rules of thumb for long polymers at neutral pH; the 33 for ssDNA is the convention NanoDrop instruments apply, from 10 mM⁻¹ cm⁻¹ per nucleotide, while Cavaluzzi and Borer measured 38 for non-repetitive ssDNA of 40–80% GC and one Thermo Fisher reference page prints 37. Oligonucleotides vary by sequence, from about 25 to 38 in the examples Thermo Fisher gives: use the extinction coefficient for the sequence and any modifications, and do not read A260/A280 or A260/A230 as purity for an oligonucleotide.
- Ultraviolet absorbance at 260 nm cannot distinguish intact double-stranded DNA from degraded RNA, free dNTPs, phenol contamination or single-stranded carrier nucleic acids.
- Microvolume spectrophotometers often report A260 already normalised to a 1.0 cm equivalent path; dividing by the physical 0.1 cm or 0.05 cm gap a second time inflates the concentration ten- to twenty-fold.
Sources and provenance
- Calculator inputs and independent arithmetic verificationOriginal synthetic inputs; see individual fixture notices
- A260 rule-of-thumb factors and their sourcesOriginal method notes
The 50 and 40 factors were read on a Thermo Fisher technical page on 6 October 2026; the 33 for ssDNA on the NanoDrop One FAQ and technical note on 17 October 2026.
- Cavaluzzi and Borer: revised UV extinction coefficients for nucleoside-5′-monophosphates and unpaired DNA and RNACited method; the 33 convention ("Method 1") and the measured 38 for non-repetitive ssDNA
- Thermo Fisher: accurate oligonucleotide quantification with NanoDrop spectrophotometers (MCS-TN1315-EN)Vendor technical note; the general ssDNA factor of 33 and oligo-specific factors, read 17 October 2026
- Wilfinger, Mackey and Chomczynski: effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purityCited method; pH dependence of A260/A280 ratios
Technical questions
Why is the dsDNA factor (50 µg/mL per A260 unit) higher than the RNA factor (40 µg/mL)?
Double-stranded DNA absorbs less 260 nm light per gram than single-stranded RNA or DNA because base stacking in the double helix reduces absorbance (the hypochromic effect). Because the specific absorbance sits in the denominator of the Beer–Lambert conversion, a lower absorbance per gram means a larger mass concentration (50 rather than 40 µg/mL) gives an absorbance of 1.0.
Why does measuring RNA in unbuffered water give a lower A260/A280 ratio than measuring in TE buffer?
Unbuffered nuclease-free water is often slightly acidic because it absorbs carbon dioxide. Wilfinger, Mackey and Chomczynski showed that the A260/A280 ratio of the same nucleic acid is lower in acidic, low-ionic-strength solution than in weakly alkaline buffer, so a ratio measured in water understates purity relative to one measured in Tris-EDTA. Blank and dilute in the same buffered solution when comparing ratios.
When should I use a fluorometric dye assay instead of A260 absorbance?
When preparing next-generation sequencing libraries, low-yield ChIP eluates or samples where genomic DNA and RNA might co-purify, intercalating fluorescent dyes selective for dsDNA or ssRNA avoid the free-nucleotide and phenol interference that inflates UV A260 readings.
Connected workflows
- Protein A280 concentration calculation
Compare nucleic acid A260 factors with sequence-derived protein A280 extinction.
- GenBank and FASTA plasmid mapping
Inspect the quantified plasmid vector sequence and feature map.
- qPCR master mix and dead-volume calculation
Use accurately quantified cDNA and template stocks in master-mix planning.