DNA Concentration Calculator

UV spectrophotometry exploits the intrinsic optical properties of aromatic molecules in nucleic acids. The purine bases (adenine and guanine) and pyrimidine bases (cytosine, thymine, and uracil) contain conjugated π-electron systems that absorb ultraviolet light. This absorption reaches maximum intensity at approximately 260 nanometers, creating a characteristic absorption spectrum that distinguishes nucleic acids from other biological macromolecules. Proteins absorb maximally at 280nm due to aromatic amino acids (tryptophan and tyrosine), while organic contaminants typically absorb around 230nm.

The Beer-Lambert Law forms the mathematical foundation for spectrophotometric quantification. This law states that absorbance (A) is directly proportional to the concentration (c) of the absorbing species, the pathlength (l) through which light travels, and the molar extinction coefficient (ε) of the molecule: A = ε × l × c. For practical DNA quantification, this relationship is simplified using empirically determined conversion factors that account for the average extinction coefficients of different nucleic acid types. Double-stranded DNA has a conversion factor of 50 μg/mL per absorbance unit, meaning an A₂₆₀ of 1.0 in a 1 cm cuvette corresponds to 50 μg/mL concentration.

The structural differences between nucleic acid types necessitate different conversion factors. Double-stranded DNA (dsDNA) exhibits hypochromicity—reduced absorbance compared to single-stranded forms—due to base stacking interactions that shield chromophores from solvent. This π-π stacking between adjacent base pairs reduces the extinction coefficient by approximately 40% compared to denatured DNA. Consequently, dsDNA uses a 50 μg/mL conversion factor, while single-stranded DNA (ssDNA) uses 33 μg/mL, and RNA uses 40 μg/mL. RNA's intermediate value reflects its tendency to form secondary structures that partially stack bases while maintaining primarily single-stranded character.

Oligonucleotide quantification requires sequence-specific calculations because short nucleic acids don't conform to the statistical averaging underlying standard conversion factors. Each oligonucleotide has a unique molar extinction coefficient calculated by summing nearest-neighbor contributions from each dinucleotide in the sequence. Online calculators and oligonucleotide suppliers typically provide these extinction coefficients. The concentration calculation then uses the formula: C = (A₂₆₀ × MW × DF) / (ε₂₆₀ × l), where MW is molecular weight and ε₂₆₀ is the extinction coefficient at 260nm.

Absorbance ratio analysis provides crucial quality metrics beyond concentration. The 260/280 ratio indicates protein contamination because proteins absorb strongly at 280nm while nucleic acids absorb weakly at this wavelength. Pure DNA exhibits a 260/280 ratio around 1.8, while pure RNA shows approximately 2.0. Ratios significantly below these values suggest protein contamination from incomplete phenol extraction or co-purification of proteins. The 260/230 ratio assesses contamination by phenol, guanidinium salts, and other organic compounds that absorb near 230nm. Pure nucleic acids show 260/230 ratios between 2.0-2.2, with lower values indicating residual contaminants that may inhibit enzymatic reactions.

Several factors affect measurement accuracy and must be controlled in experimental practice. Sample pH significantly impacts absorbance; nucleic acids should be dissolved in neutral pH buffers (pH 7-8) because extreme pH values denature the double helix and alter chromophore ionization states. Temperature effects are generally minimal near room temperature but become significant if samples are heated above 70°C (denaturation) or cooled near 0°C (conformational changes). Ionic strength influences hyperchromicity slightly but is usually standardized by using TE buffer (10mM Tris, 1mM EDTA) or similar nucleic acid storage buffers for both samples and blanks.