How DNA Concentration Is Calculated
Spectrophotometers (such as a NanoDrop) measure how much ultraviolet light a sample absorbs at a wavelength of 260 nanometres, the point at which nucleic acids absorb most strongly. This reading, called A260, is converted into a concentration using the Beer-Lambert law. For double-stranded DNA, an A260 reading of 1.0 corresponds to a concentration of approximately 50 ng/uL when measured in a standard 1 cm path length cuvette. The formula is:
Concentration (ng/uL) = A260 x extinction coefficient x dilution factor
The extinction coefficient differs by nucleic acid type: 50 ng/uL per A260 unit for double-stranded DNA, 33 ng/uL per A260 unit for single-stranded DNA, and 40 ng/uL per A260 unit for RNA. If your sample was diluted before being measured (for example, 1:10 in water), multiply the result by the dilution factor to get the concentration of the original, undiluted sample.
Assessing Purity with A260/A280
The A260/A280 ratio compares absorbance from nucleic acids (260 nm) against absorbance from proteins (280 nm, largely from aromatic amino acids). A ratio around 1.8 is considered good quality for DNA, while a ratio around 2.0 is expected for pure RNA. A ratio noticeably below 1.8 suggests protein or phenol contamination from the extraction process, and a ratio well above 2.0 can indicate RNA contamination in a DNA prep. This ratio is a useful quality check, but it is only one indicator; gel electrophoresis or fluorometric quantification (such as Qubit) are more reliable for low-concentration samples, since UV absorbance is less accurate below about 10 ng/uL.
Working Out Molarity
For applications like PCR primer design or library prep, concentration is sometimes needed in molar terms (nM) rather than mass terms (ng/uL). This requires knowing the average molecular weight of a base pair (approximately 650 g/mol per bp for dsDNA). The conversion is:
Molarity (nM) = (concentration in ng/uL x 10^6) / (average bp length x 650)
This calculator estimates molarity using this standard approximation once you enter the average fragment or amplicon length in base pairs.
Planning a Dilution
To dilute a concentrated stock down to a target working concentration, use the standard dilution equation C1V1 = C2V2, where C1 and V1 are the concentration and volume of your stock, and C2 and V2 are the concentration and volume you want to end up with. Rearranged to solve for the volume of stock needed: V1 = (C2 x V2) / C1. The remaining volume (V2 minus V1) is made up with diluent, typically nuclease-free water or TE buffer.
Worked Example
An A260 reading of 0.400 measured neat (dilution factor 1) for dsDNA gives a concentration of 0.400 x 50 x 1 = 20 ng/uL. In a 50 uL sample, that is a total yield of 20 x 50 = 1,000 ng (1 ug) of DNA. With an A280 of 0.222, the A260/A280 ratio is 0.400 / 0.222 = 1.80, indicating good purity. To dilute this stock down to a 10 ng/uL working concentration in a final volume of 50 uL, you need V1 = (10 x 50) / 20 = 25 uL of stock, topped up with 25 uL of diluent.
Related Calculators
Sources: Beer-Lambert law and standard dsDNA extinction coefficients as used in NanoDrop and general molecular biology laboratory protocols. Average base pair molecular weight (650 g/mol) is a standard approximation used in molecular biology for double-stranded DNA.
This calculator uses the standard Beer-Lambert approximation for nucleic acid quantification by UV absorbance. Actual concentration accuracy depends on your spectrophotometer's calibration, sample purity, and path length correction. Fluorometric methods (such as Qubit or PicoGreen) are more accurate for low concentration or highly pure samples. This tool is for laboratory planning purposes and does not replace calibrated instrument software.
