Estimating how much DNA or RNA is in a sample is one of the first checks in almost every molecular biology workflow ā before cloning, sequencing, PCR, or transfection. Modern spectrophotometers and NanoDrop instruments make this fast, but interpreting the numbers correctly still depends on understanding what A260, the conversion factor, and the 260/280 purity ratio actually mean.
Why A260 measures nucleic-acid concentration
DNA and RNA absorb ultraviolet light most strongly around 260 nanometers, largely due to the aromatic rings in their nucleotide bases. Under the Beer-Lambert law, absorbance is directly proportional to concentration for a fixed path length, so a spectrophotometer reading at 260 nm (A260) can be converted into a concentration once you know the right proportionality constant for the type of nucleic acid you're measuring.
Why the factor differs by nucleic-acid type
The conversion factor ā 50 Āµg/mL per A260 unit for double-stranded DNA, 33 Āµg/mL for single-stranded DNA, and 40 Āµg/mL for RNA ā differs because these molecules absorb 260 nm light slightly differently per unit mass. Double helix base-stacking in dsDNA reduces its absorbance somewhat compared to unstructured single strands, which is why ssDNA and RNA (both typically less base-paired in solution) have lower per-mass factors. Using the wrong factor for your sample type is one of the most common sources of an inaccurate concentration estimate.
Reading the 260/280 purity ratio
Proteins absorb UV light most strongly around 280 nm because of aromatic amino acids like tryptophan and tyrosine, while nucleic acids absorb most strongly at 260 nm. Comparing the two absorbances (A260/A280) therefore gives a quick estimate of how much protein contamination remains in an extracted sample. A ratio near 1.8 is considered pure double-stranded DNA; a ratio near 2.0 is considered pure RNA. Ratios noticeably below these values usually point to leftover protein or phenol from the extraction process, while ratios noticeably above can indicate RNA contamination in a DNA prep or degraded sample.
Why dilution matters for accurate readings
Spectrophotometer absorbance readings are only reliably linear with concentration up to roughly an A260 of 1.0ā2.0, depending on the instrument. Samples more concentrated than that should be diluted before reading, then the diluted reading's calculated concentration multiplied by the dilution factor to recover the original stock's concentration ā which is exactly what this calculator's Dilution tab automates.
Limitations of A260-based estimates
A260/A280-based estimates are fast and convenient but are less precise than fluorescence-based quantification methods (like PicoGreen or Qubit assays), which specifically bind double-stranded DNA and are far less sensitive to contaminating RNA, free nucleotides, or protein. For applications with tight quantification requirements ā like next-generation sequencing library prep ā labs often confirm a NanoDrop-based estimate with a fluorescence-based assay before proceeding.