DNA quantification & QC
Microvolume absorbance: 1–2 µL on a pedestal gives a concentration and the A260/A280, A260/A230 and A320 readings in seconds, with the sample recoverable. The fastest way to see whether a prep is contaminated, and the least reliable way to know how much DNA is in it.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
NanoDrop
Microvolume absorbance: 1–2 µL on a pedestal gives a concentration and the A260/A280, A260/A230 and A320 readings in seconds, with the sample recoverable. The fastest way to see whether a prep is contaminated, and the least reliable way to know how much DNA is in it.
How it differs from the standard method
- Measures every base in the tube, so RNA, free nucleotides and single-stranded DNA are all counted as DNA.
- Practical floor around 2 ng/µL: below that the absorbance is the same size as the instrument's baseline noise.
- The conversion factor is a setting, not a measurement (50, 40 or 33 ng/µL per absorbance unit) and the wrong one is a silent error.
- The purity ratios are its real value: A260/A230 is the only routine reading that sees guanidine, phenol and carbohydrate.
- A 1 µL drop is the least reproducible part of the measurement, so read three times and look at the spread.
1Three questions, three different measurements
"How much DNA have I got?" is really three questions, and no single instrument answers all of them. How much double-stranded DNA is there, by mass?
Counts bases
Fast, needs 1–2 µL, consumes nothing, and gives purity ratios. But it counts every base in the tube (dsDNA, ssDNA, RNA, free nucleotides) and has a floor around 2 ng/µL.
Counts intact duplex
An intercalating dye fluoresces only when bound inside double-stranded DNA. Accurate from picograms up, blind to RNA, and blind to everything else too: no ratios, no size.
Counts and measures
Separates the sample by size against a ladder and quantifies by fluorescence. Gives peak size, N50, an integrity score and molarity, from a few µL at a few pounds a lane.
2Why absorbance and a dye disagree
At 260 nm every nucleotide base absorbs, whether it sits in a duplex, in a single strand, in RNA or free in solution. The conversion factors differ (about 50 ng/µL per absorbance unit for dsDNA, 40 for RNA, 33 for single-stranded DNA) because stacked bases in a helix absorb slightly less per base than free ones.
3Reading the purity ratios
- A260/A280 ≈ 1.8–1.9
- Compares the base absorbance at 260 nm with the aromatic amino-acid absorbance at 280 nm. Protein carry-over pulls it below 1.75; RNA or free nucleotides push it above 2.0. It is blind to salt, blind to degradation, and nearly blind to RNA.
- A260/A230 ≈ 2.0–2.2
- The ratio that actually sees contamination. Guanidinium salts, phenol, carbohydrates and EDTA all absorb steeply below 240 nm. A value under about 1.8 means something is in the tube that a polymerase or ligase will notice, and A260/A280 can look perfect at the same time.
- A320 should be zero
- Nothing in a clean nucleic-acid sample absorbs at 320 nm, so anything there is particulate scattering. It is the quickest sanity check on a spectrum and the usual sign of a sample that needs a spin or a filter.
4Why a concentration is not a molarity
Ligations, library pooling, loading a sequencer and dosing a transfection are all specified in moles, not mass, because what matters is the number of molecules. Converting needs the fragment length: nM = ng/µL × 10⁶ ÷ (660 × bp), where 660 g/mol is the average molar mass of a base pair.
5Integrity, and why it is invisible to the other two
A DNA prep that has been nicked and sheared into two-kilobase fragments has exactly the same mass, the same absorbance ratios and the same dye reading as the intact prep it came from. Degradation changes the size distribution and nothing else that either method measures.
Key terms
Practise these as flashcards →ng/µL per absorbance unit at 260 nm over a 1 cm path: about 50 for dsDNA, 40 for RNA, 33 for ssDNA. Choosing the wrong one is a silent systematic error of up to a third.
A dye such as the Qubit or PicoGreen chemistry that fluoresces strongly only when stacked between base pairs, making the assay specific for intact double-stranded DNA.
Quoted as mass of DNA in the assay tube (roughly 0.2–120 ng high-sensitivity, 2–1200 ng broad-range), because the limit is the amount of dye available to bind.
DNA Integrity Number, 1–10, summarising how much of a sizing trace sits at high molecular weight. The DNA analogue of RIN, the RNA Integrity Number used for RNA on the same instruments.
The fragment length above which half the mass of the sample lies. More useful than a mean for a skewed distribution, and the number long-read protocols specify.
nM = ng/µL × 10⁶ ÷ (660 × bp). The 660 g/mol per base pair is an average; the bp has to come from a size measurement.
About 2 ng/µL for absorbance however carefully you read it, because the signal becomes the same size as the baseline noise; picograms per µL for a fluorescent dye.
The size window between a sizing assay's lower and upper markers. Material outside it is included in the concentration but cannot be sized.
When to use it
- Before a library prep, where the input is specified as a mass of double-stranded DNA and an error of two-fold in that number changes the library yield and the duplicate rate.
- Before pooling libraries or loading a sequencer, where the specification is in nM and you need a concentration and a fragment size together.
- Before a long-read or optical-mapping run, where the fragment size distribution rather than the concentration decides whether the sample is usable at all.
- After any prep where carry-over is plausible, to see whether the eluate will inhibit the enzyme you are about to add to it.
- On archived, low-input or cell-free samples, where absorbance is below its limit of detection and only a fluorescent dye can give a number.
Compared with alternatives
Prefer it when the question is about the measurement itself, blanking, path length, linear range, stray light, the shape of the spectrum. This page is about which number to act on once you have it.
The step that produces the samples. QC results feed straight back into it: a low A260/A230 means the wash step, a low DIN means the handling.
The other common source of samples, where residual RNA is the usual reason absorbance and a dye disagree.
The cheap version of the sizing measurement: it shows integrity and a genomic smear for pennies, but band intensity is at best a two-fold estimate of mass.
Where the molarity calculation is actually used, and the reason a concentration without a fragment size is not enough.
The same instruments and the same logic applied to RNA, where RIN has been the standard integrity measure for much longer.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Microvolume absorbance: 1–2 µL on a pedestal gives a concentration and the A260/A280, A260/A230 and A320 readings in seconds, with the sample recoverable. The fastest way to see whether a prep is contaminated, and the least reliable way to know how much DNA is in it.
- Measures every base in the tube, so RNA, free nucleotides and single-stranded DNA are all counted as DNA.
- Practical floor around 2 ng/µL: below that the absorbance is the same size as the instrument's baseline noise.
- The conversion factor is a setting, not a measurement (50, 40 or 33 ng/µL per absorbance unit) and the wrong one is a silent error.
- The purity ratios are its real value: A260/A230 is the only routine reading that sees guanidine, phenol and carbohydrate.
- A 1 µL drop is the least reproducible part of the measurement, so read three times and look at the spread.