DNA Concentration Calculator (A260 Spectrophotometry)

Formula

How this is calculated

The Beer-Lambert law relates absorbance to concentration: A = ε × c × l, where ε is the molar extinction coefficient, c is concentration, and l is the path length (typically 1 cm in a cuvette or ~0.1 mm in a NanoDrop). For nucleic acids, empirically derived extinction coefficients per A260 unit (at 1 cm path length) are: 50 µg/mL for double-stranded DNA, 33 µg/mL for single-stranded DNA and oligonucleotides, and 40 µg/mL for single-stranded RNA. Multiplying A260 by the appropriate coefficient and by the dilution factor gives concentration in µg/mL, which equals ng/µL (useful since most molecular biology volumes are in microlitres).

The A260/A280 ratio assesses sample purity. Pure dsDNA has a ratio of about 1.8; pure RNA is around 2.0. Ratios below ~1.7 suggest contamination with protein (which absorbs strongly at 280 nm due to aromatic amino acids) or phenol carryover from extraction. Values above ~2.1 may indicate RNA contamination in a DNA preparation. The A260/A230 ratio (not shown) further flags salt, EDTA or solvent contamination — values below 1.7 indicate impurities absorbing at 230 nm.

Total mass yield (µg) = concentration (µg/mL) × volume (mL), where 1 µL = 0.001 mL. The path-length correction factor used by NanoDrop and similar micro-volume instruments is handled internally by those devices — enter the corrected A260 reading displayed by the instrument, not the raw detector signal.