UV spectrophotometry is the default quantification method in most molecular biology labs. It is fast, it requires no additional reagents, and the NanoDrop is on every bench. It is also unreliable for Exymes extracts, and the reason is straightforward once you know what to look for.

Exymes extracts do not require purification before downstream use. The extraction buffers and the amino acids released during protease digestion remain in solution alongside the DNA. Several of those compounds, including aromatic amino acids such as phenylalanine, absorb UV light in the same range as nucleic acids. The A260 reading picks them up. The reported concentration goes up. The actual DNA concentration does not.

In a direct comparison across buccal, plant, and bacterial extracts, UV spectrophotometry dramatically overestimated DNA concentration relative to both fluorometric assays (Qubit, PicoGreen, iQuant) and qPCR. The fluorometric and qPCR methods agreed closely with each other. The spectrophotometer did not agree with either.

This matters when downstream steps require a specific DNA input: PCR optimisation, library preparation, or any application where knowing the true template amount affects the result. Overloading with excess template creates its own problems.

The recommendation from EXAPN016 is consistent: use fluorometric quantification or qPCR for Exymes extracts. Keep the NanoDrop for samples that have been through a purification step. For crude enzymatic extracts, it is the wrong tool for this particular job.