DNA Concentration Calculator
The DNA concentration calculator applies concentration = A260 × factor × dilution ÷ path length, with 50 for dsDNA, 33 for ssDNA and 40 for RNA. It also reads the purity ratios and works out how many molecules that is.
Concentration
- Concentration 375 µg/mL, which is the same number as ng/µL. In the measured, diluted solution it was 37.5 ng/µL.
- 375 ng/µL
- Working Beer-Lambert written for nucleic acids: concentration = absorbance × factor ÷ path length, then multiplied back up by the dilution. The factor is the concentration that gives an A260 of 1.0 in a 1 cm path.
- 0.75 × 50 µg/mL ÷ 1 cm × 10 = 375 µg/mL
- Conversion factor for dsDNA A convention, not a constant: the factor assumes typical base composition and a long molecule. Thermo Fisher's NanoDrop documentation uses 50 for dsDNA, 33 for ssDNA and 40 for RNA, the figures most labs use.
- 50 µg/mL per A260 unit
- Total in the sample 375 ng/µL × 50 µL. This is the yield of the prep, if the reading is representative.
- 18.8 µg
- A260/A280 About 1.8, the value generally accepted as pure for DNA. No sign of protein or phenol carry-over.
- 1.88
- A260/A230 In the 2.0-2.2 range commonly expected for pure nucleic acid.
- 2.08
- Molecular weight of 3,000 bp 3,000 × 661.86 g/mol per base pair (sodium salt), summed from IUPAC atomic weights. End groups change it by well under 100 g/mol, which is negligible for anything longer than a primer.
- 1,985,589 g/mol
- Copies in 10 ng copies = mass × Avogadro ÷ molecular weight = 10 × 10^-9 g × 6.02214076 × 10^23 ÷ 1,985,589 g/mol. Avogadro's constant has been exact since the 2019 SI redefinition.
- 3.03 × 10^9
- Copies per µL of the sample From the measured 375 ng/µL. The starting point for a qPCR standard curve.
- 1.14 × 10^11
- Molar concentration 375 ng/µL ÷ 1,985,589 g/mol. Molecules, not ends or bases: for a ligation, ends are twice this for linear dsDNA.
- 189 nM
| Dilution | Concentration | Copies per µL |
|---|---|---|
| neat | 375 ng/µL | 1.14 × 10^11 |
| 1:10 | 37.5 ng/µL | 1.14 × 10^10 |
| 1:100 | 3.75 ng/µL | 1.14 × 10^9 |
| 1:1,000 | 0.375 ng/µL | 1.14 × 10^8 |
| 1:10,000 | 37.5 pg/µL | 1.14 × 10^7 |
| 1:100,000 | 3.75 pg/µL | 1.14 × 10^6 |
| 1:1,000,000 | 0.375 pg/µL | 1.14 × 10^5 |
| 1:10,000,000 | 0.0375 pg/µL | 1.14 × 10^4 |
| 1:100,000,000 | 0.00375 pg/µL | 1.14 × 10^3 |
- A260 counts every nucleotide in the tube: DNA, RNA, free nucleotides and degraded fragments alike. A DNA prep with RNA in it reads high. A dye-based assay that binds only dsDNA (or only RNA) is the way to measure one in the presence of the other.
- Very low and very high absorbances are the least reliable. Dilute or concentrate the sample to bring the reading into your instrument's stated linear range, and enter the dilution.
- Blank against the same buffer the sample is in, at the same pH and similar ionic strength. Blanking with water for a sample dissolved in TE, for example, gives a misleading 260/230.
How the DNA Concentration Calculator works
The formula is Beer-Lambert written for nucleic acids:
concentration (µg/mL) = A260 × conversion factor ÷ path length (cm) × dilution factor
with a conversion factor of 50 for double-stranded DNA, 33 for single-stranded DNA and 40 for RNA. µg/mL and ng/µL are the same number, so the result reads directly as ng/µL.
A worked example, the one above: a plasmid prep diluted 1 in 10 reads A260 = 0.75 in a 1 cm cuvette. 0.75 × 50 = 37.5 µg/mL in the cuvette, and × 10 for the dilution gives 375 ng/µL in the tube. In 50 µL that is 18.75 µg of DNA.
Where 50, 33 and 40 come from. Each is the concentration that gives an absorbance of 1.0 at 260 nm in a 1 cm path. They are conventions rather than constants: the true figure depends on base composition and on how much the bases stack, which is why double-stranded DNA (stacked, absorbing less per base) gets a higher factor than single strands. These are the values Thermo Fisher's NanoDrop documentation uses, and they are what nearly every lab means by "an A260 of 1 is 50 µg/mL". For short oligonucleotides the general 33 can be off by more than 20%, because a short sequence's composition is far from average; use the factor or extinction coefficient on the supplier's data sheet.
Path length. A standard cuvette is 1 cm. Microvolume instruments such as the NanoDrop measure over a much shorter path but report absorbance normalized to 1 cm, so for their readings the path length here stays at 1. Enter a different path only for a raw reading from a non-standard cell.
Purity ratios, in plain words. Proteins absorb most at 280 nm and many extraction reagents absorb at 230 nm, so the ratios of A260 to those readings flag carry-over. An A260/A280 of about 1.8 is generally accepted as pure DNA, and about 2.0 as pure RNA. Appreciably lower suggests protein, phenol or another reagent. A DNA prep at 2.0 is not contaminated with protein, but may contain RNA, which A260 cannot tell from DNA. A260/A230 is commonly expected at 2.0-2.2; lower points to guanidine salts from a spin column, phenol, carbohydrate or a glycogen carrier, and those also inflate A260 and so the concentration. An unusually high 260/230 is usually a blanking problem rather than the sample.
Copy number. For a qPCR standard or a transfection you need molecules, not nanograms:
copies = mass (g) × Avogadro constant ÷ (length × mass per base pair)
Avogadro's constant is exactly 6.02214076 × 10^23 per mole since the 2019 SI redefinition. The mass per base pair is computed here from the IUPAC atomic weights: 661.9 g/mol for a sodium-salt base pair (DNA in buffer) and 617.9 for the free acid. Many protocols use a round 650 or 660, within 2% of the computed figure and well inside the error of the A260 reading. In the example, 10 ng of the 3,000 bp plasmid is 10 × 10^-9 × 6.022 × 10^23 ÷ (3,000 × 661.9) = 3.03 × 10^9 copies.
What this calculator does not do: correct for turbidity (some instruments subtract A320 or A340), separate DNA from RNA, or account for modifications and dyes on an oligo. For those, use a fluorometric assay or the supplier's figures.
DNA Concentration Calculator FAQ
How do you calculate DNA concentration from A260?
Multiply the A260 by 50 µg/mL for double-stranded DNA (33 for single-stranded, 40 for RNA), divide by the path length in cm, and multiply by any dilution. An A260 of 0.75 at a 1 in 10 dilution is 0.75 × 50 × 10 = 375 µg/mL, which is 375 ng/µL.
What is a good 260/280 ratio?
About 1.8 is generally accepted as pure for DNA and about 2.0 for RNA. A ratio well below that suggests protein or phenol carry-over. A DNA sample reading near 2.0 is not protein-contaminated, but may contain RNA.
What does a low 260/230 ratio mean?
Something in the sample absorbs at 230 nm. Guanidine salts from column kits, phenol, carbohydrates and glycogen are the common causes. They also add to the A260, so a sample with a low 260/230 usually has less nucleic acid than the concentration suggests.
How do I calculate DNA copy number?
Copies = mass in grams × 6.02214076 × 10^23 ÷ (length in bp × mass per base pair). With a sodium-salt base pair at 661.9 g/mol, 10 ng of a 3,000 bp plasmid is about 3.0 × 10^9 copies. Use about 330 g/mol per nucleotide for single strands.
Is ng/µL the same as µg/mL?
Yes. Both are 10^-6 grams per milliliter, so the number is identical. Spectrophotometer software tends to report ng/µL; older protocols write µg/mL.