Nucleic Acid Calculator | DNA & RNA Concentration Tool

Several factors can affect purity ratios: 1) Protein contamination typically lowers the A260/A280 ratio below 1.8. 2) Residual phenol from extraction can artificially increase the ratio above 2.0. 3) pH significantly affects readings—samples in water (more acidic) read lower than in TE buffer (more basic). 4) Very dilute samples give unreliable ratios due to the detection limit of spectrophotometers. 5) Salt concentration affects readings—high salt can shift absorbance curves. If your ratio is outside expected ranges, consider re-extraction or additional purification steps before proceeding with sensitive applications.

Spectrophotometric (absorbance-based) methods and fluorometric methods each have distinct advantages. Spectrophotometry is simpler and requires no reagents, but measures all nucleic acids indiscriminately, including degraded DNA/RNA and free nucleotides. It's less sensitive, requiring ~2 ng/μL minimum concentration. Fluorometric methods (like Qubit or PicoGreen) selectively measure intact nucleic acids by using dyes that fluoresce when bound to double-stranded molecules. They're much more sensitive (detecting as low as 0.1 ng/μL) and unaffected by most contaminants. For precise work with limited or potentially contaminated samples, fluorometric methods are generally more accurate.

Extinction coefficients differ between dsDNA, ssDNA, and RNA due to their distinct structural properties. In double-stranded DNA, some bases are sheltered within the helix structure (hypochromicity), reducing their ability to absorb UV light compared to the same bases in single-stranded form. This is why dsDNA has a lower extinction coefficient (50) than ssDNA (33). RNA contains uracil instead of thymine and typically exists as a single strand with complex secondary structures, giving it an intermediate extinction coefficient (40). These differences in molecular arrangement and base composition directly affect how efficiently the molecules absorb light at 260 nm, necessitating different conversion factors when calculating concentration from absorbance.