Oligo and DNA/RNA Conversion Cheat Sheet
A bench reference for the conversions you reach for when quantifying nucleic acids: turning an A260 reading into a concentration, checking purity with A260/A280 and A260/A230 ratios, converting between ng and pmol, estimating copy number, and resuspending a freshly synthesized oligo to a target molarity. Exact figures depend on the sequence — see the note below.
Reading A260 / quantifying by UV
A spectrophotometer reads absorbance at 260 nm. One A260 unit (1 cm path length) corresponds to a different concentration depending on the molecule, because single- and double-stranded nucleic acids absorb differently.
| Nucleic acid | Concentration per A260 unit |
|---|---|
| dsDNA | ≈ 50 µg/mL per A260 |
| ssDNA (incl. oligos) | ≈ 33 µg/mL per A260 |
| ssRNA | ≈ 40 µg/mL per A260 |
The single-stranded oligo factor is sequence-dependent — the true value comes from nearest-neighbor extinction coefficients, so 33 µg/mL per A260 is a rule of thumb. Verify for your sequence when precision matters.
Purity ratios (A260/A280, A260/A230)
Absorbance ratios flag contaminants. A260/A280 catches protein and phenol; A260/A230 catches salts, solvents and carbohydrate carryover. They are diagnostic, not exact — values vary by source and instrument.
| Ratio | Expected (pure) | Low value suggests |
|---|---|---|
| A260/A280 | ~1.8 = pure DNA; ~2.0 = pure RNA | lower suggests protein / phenol contamination |
| A260/A230 | ~2.0–2.2 expected | lower suggests guanidine, phenol, EDTA or carbohydrate carryover |
ng ↔ pmol and copy number
When you only know the length, use these average molecular weights — the same values SeqBench's DNA Molarity tool uses.
| Nucleic acid | Average molecular weight |
|---|---|
| dsDNA | ≈ 650 g/mol per base pair |
| ssDNA | ≈ 330 g/mol per nucleotide |
| ssRNA | ≈ 340 g/mol per nucleotide |
The core conversions:
- pmol = (mass in ng × 1000) / molar mass (g/mol)
- molar mass of dsDNA ≈ length(bp) × 650 → pmol dsDNA ≈ ng × 1000 / (bp × 650)
- copies = moles × Avogadro (6.022 × 10²³); moles = (ng × 1e-9) / molar mass(g/mol)
- concentration: 1 pmol/µL = 1 µM; multiply by 1000 for nM
Worked example. 1 µg (1000 ng) of a 1 kb dsDNA fragment: 1000 × 1000 / (1000 × 650) ≈ 1.54 pmol.
Resuspending an oligo to a target concentration
Synthesized oligos ship dry with the yield given in nmol on the spec sheet. Because nmol/µL equals mM, you can hit a round stock molarity just by choosing the right water volume.
| Target stock | Water to add | Example |
|---|---|---|
| 100 µM | add (nmol × 10) µL water | 20 nmol → 200 µL |
| 1 mM (1000 µM) | add (nmol × 1) µL water | 20 nmol → 20 µL |
The logic: nmol/µL = mM. Dissolving an oligo's nmol amount in (nmol × 10) µL gives 0.1 mM = 100 µM; in (nmol × 1) µL it gives 1 mM = 1000 µM.
These average-mass shortcuts are close enough for routine work, but exact values need the actual sequence (nearest-neighbor). The DNA Molarity tool does the conversion precisely from your sequence.
Frequently asked questions
How do I convert A260 to concentration?
Multiply the A260 reading (at 1 cm path length) by the conversion factor for your molecule: about 50 µg/mL per A260 unit for dsDNA, 33 µg/mL for single-stranded DNA and oligos, and 40 µg/mL for ssRNA. So an A260 of 0.5 for dsDNA is roughly 25 µg/mL. The single-stranded factors are rules of thumb and are sequence-dependent.
What does an A260/A280 of 1.8 mean?
An A260/A280 ratio of about 1.8 indicates pure DNA; about 2.0 indicates pure RNA. A noticeably lower ratio suggests protein or phenol contamination, since those absorb strongly at 280 nm.
How do I convert ng to pmol of DNA?
Use pmol = (mass in ng × 1000) / molar mass in g/mol. For dsDNA the molar mass is about length(bp) × 650, so pmol ≈ ng × 1000 / (bp × 650). For example, 1 µg (1000 ng) of a 1 kb dsDNA fragment is about 1000 × 1000 / (1000 × 650) ≈ 1.54 pmol.
How much water do I add to resuspend an oligo to 100 µM?
Add (nmol × 10) µL of water — the nmol figure is on the synthesis spec sheet. For example, a 20 nmol oligo needs 200 µL to reach 100 µM. For a 1 mM (1000 µM) stock, add (nmol × 1) µL instead, so 20 nmol needs 20 µL.
How do I calculate copy number?
Convert mass to moles, then multiply by Avogadro's number: moles = (ng × 1e-9) / molar mass(g/mol), and copies = moles × 6.022 × 10²³. Knowing the fragment length lets you estimate molar mass as bp × 650 for dsDNA.
See also
Sources
- 1NanoDrop 2000/2000c Spectrophotometer V1.0 User Manual, 'Nucleic Acid Calculations'Thermo Fisher Scientific · 2009All three rows of the A260 table, exactly as printed. Fetched the official Thermo-hosted PDF and read the section: 'The generally accepted extinction coefficients for nucleic acids are: Double-stranded DNA: 50 ng-cm/uL; Single-stranded DNA: 33 ng-cm/uL; RNA: 40 ng-cm/uL', used in c = (A * e)/b with absorbance normalised to a 1.0 cm path — which is also the source of the page's '1 cm path length' qualifier. The instrument's sample-type names are literally DNA-50, ssDNA-33 and RNA-40. Its own wording, 'generally accepted', supports the page's rule-of-thumb framing.
- 2Effect of pH and Ionic Strength on the Spectrophotometric Assessment of Nucleic Acid PurityWilfinger WW, Mackey K, Chomczynski P · BioTechniques 22(3):474-481 (PMID 9067025) · 1997The primary study behind the A260/A280 row — it is the experimental basis for treating ~1.8 (DNA) and ~2.0 (RNA) as the 'pure' ratio, and for the caveat in the page's intro that these 'are diagnostic, not exact - values vary by source and instrument': the paper shows the RNA A260/280 ratio moves from ~1.5 to ~2.0 purely by shifting the water from pH ~5.4 to pH 7.5-8.5, with no change in purity. The DOI resolves (302 to Taylor & Francis) but the publisher returns 403 to automated fetching, so I confirmed the citation two other ways: a search hit on PubMed/T&F, and the reference list of Thermo Technical Note 52646 below, which I did fetch and which cites this paper as its sole reference.
- 3Assessment of Nucleic Acid Purity (Technical Note 52646)Matlock B · Thermo Fisher Scientific NanoDrop technical note · 2015Every cell of the PURITY table, in the page's own wording. Fetched and read in full: 'A ratio of ~1.8 is generally accepted as "pure" for DNA; a ratio of ~2.0 is generally accepted as "pure" for RNA'; 'Expected 260/230 values are commonly in the range of 2.0-2.2'; 'A low A260/A280 ratio may be caused by: Residual phenol or other reagent associated with the extraction protocol'; and for A260/A230, 'Carbohydrate carryover... Residual phenol... Residual guanidine (often used in column based kits). Glycogen used for precipitation.' One item on SeqBench's A260/A230 row, EDTA, is not in that bullet list — the bulletin covers it obliquely ('Using water for the Blank measurement for samples dissolved in TE may result in low 260/230 ratios'), so EDTA is supported as a blanking/buffer artefact rather than as a listed contaminant.
- 4Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNACavaluzzi MJ, Borer PN · Nucleic Acids Research 32(1):e13 · 2004The footnote under the A260 table ('the true value comes from nearest-neighbour extinction coefficients, so 33 ug/mL per A260 is a rule of thumb') and the closing note that exact values need the actual sequence. Fetched the full text at Oxford Academic: the paper re-measures the mononucleotide extinction coefficients that all of these factors descend from, finds the older published values too large by up to 7%, and recommends 38 ug per A260 unit for non-repetitive ssDNA rather than the traditional 33 — so it substantiates the caveat in the specific direction the page needs. Pair with Tataurov AV, You Y, Owczarzy R, Biophys Chem 133:66-70 (2008), doi:10.1016/j.bpc.2007.12.004, if a nearest-neighbour-model citation is wanted specifically; I confirmed that paper's title, journal, volume, pages and DOI by search but did not fetch it (ScienceDirect abstract only).