Nucleic Acid Calculator — NanoDrop Concentration & Purity (A260/A280)
Estimate dsDNA, RNA or ssDNA concentration from A260, the absorbance path basis and dilution factor. Calculate A260/A280 and A260/A230 ratios, with reference spectral patterns to explain possible carryover.
Nucleic Acid Concentration & Absorbance Ratios
For NanoDrop nucleic-acid application readouts already normalized to 10 mm, select 10 mm even when the instrument used a shorter physical path. Choose a short path only for absorbance explicitly reported on that basis. Enter all three absorbances on the same path basis.
Use blank-corrected absorbance. A280 and A230 are optional; their ratios require positive A260 and denominator values.
Original-sample concentration estimate
42.5 ng/µL
0.043 µg/µL = 0.043 mg/mL
Total yield estimate
2.13 µg
Uses the original sample volume; volume must be positive.
A260 / A280
1.85
Typical ≈ 1.8; comparison 1.7–2.
A260 / A230
2.07
Common reference: about 2.0–2.2.
Concentration = A260 × (10 / path in mm) × 50 × dilution factor. Total yield (µg) = concentration (ng/µL) × original volume (µL) / 1000.
Comparison ranges are screening guides. Low or high ratios can reflect carryover, pH, blank mismatch or measurement noise; values within range do not prove purity or integrity. Consult the instrument range, especially at low absorbance.
Beer-Lambert Law & Extinction Coefficients
Nucleic acid quantification relies on the Beer-Lambert Law:
A is absorbance at 260 nm, b is path length (cm), and ε is the extinction coefficient. Concentration units must match ε: a molar coefficient gives c in mol/L. The empirical factors below estimate mass concentration at a 10 mm (1 cm) equivalent path:
- Double-Stranded DNA (dsDNA): 1.0 A260 = 50 µg/mL (50 ng/µL)
- Single-Stranded RNA: 1.0 A260 = 40 µg/mL (40 ng/µL)
- Single-Stranded DNA (general estimate): 1.0 A260 ≈ 33 µg/mL (33 ng/µL)
The factors 50 and 40 are also conventional estimates. Short oligos, modified nucleic acids and specific sequences require their own extinction coefficients for precise quantification. Other absorbing molecules contribute to A260, so this is a UV-based concentration estimate.
Quality Control: A260/A280 & A260/A230 Bench Criteria
The values below are approximate references, not contaminant-identification thresholds. The calculator compares dsDNA A260/A280 with 1.7–2.0, RNA with 1.9–2.2, and A260/A230 with 2.0–2.2. These comparison windows prompt review; they do not establish purity or integrity.
| Sample | A260/A280 | A260/A230 | Interpretation limits |
|---|---|---|---|
| dsDNA | ≈ 1.8 | ≈ 2.0–2.2 | Depends on buffer, pH and concentration |
| RNA | ≈ 2.0 | ≈ 2.0–2.2 | Ratios do not measure RNA integrity |
| ssDNA / oligos | Sequence and measurement dependent | No universal pass/fail threshold; use a sequence-specific extinction coefficient | |
UV absorbance patterns: DNA and possible carryover
Teaching schematic, not measured data. The DNA reference is scaled to A260 = 1; the other curves add illustrative absorber contributions to the same DNA background. Absorbers may also change A260, so the three curves are not separately normalized.
A 230 nm increase can occur with guanidine carryover; it is not a unique fingerprint. Phenol can also alter the 260–280 nm region. Compare the actual instrument scan, blank and assay results before attributing contamination.
Interpreting the plot and unusual ratios
The curves illustrate possible absorber contributions and do not change with calculator inputs. They do not replace a measured sample spectrum. A230, A260 and A280 alone cannot reconstruct a full spectrum or locate its peaks.
Low A260/A280 can reflect protein, phenol, pH or blank mismatch; low A260/A230 can reflect extraction-reagent carryover or blank mismatch. Very high ratios also warrant remeasurement, especially when the denominator is close to zero. Check the blank, instrument range and repeatability before following the extraction kit’s troubleshooting instructions.
References: Thermo Scientific NanoDrop Nucleic Acid Technical Guide · NanoDrop 2000 FAQ
Nucleic Acid Calculator Worked Lab Example: Plasmid Miniprep QC
Scenario: Miniprep Plasmid DNA Yield & Purity Assessment
An undiluted plasmid sample (dilution factor 1) gives 10 mm-equivalent NanoDrop nucleic-acid readouts of A260 = 1.62, A280 = 0.88 and A230 = 0.77. Original sample volume is 50 µL.
- 1. 10 mm-equivalent A260 = 1.62 (already normalized by the instrument)
- 2. Plasmid Concentration = 1.62 × 50 ng/µL = 81.0 ng/µL
- 3. Total Yield = 81.0 ng/µL × 50 µL = 4,050 ng = 4.05 µg
- 4. A260/A280 = 1.62 / 0.88 = 1.84 (within a common dsDNA reference range)
- 5. A260/A230 = 1.62 / 0.77 = 2.10 (within a common reference range)
If A260 = 1.62 were an unnormalized 1 mm reading, the estimate would be 810 ng/µL and 40.5 µg. Do not apply that extra tenfold correction to an already normalized 10 mm NanoDrop nucleic-acid readout.
Frequently Asked Questions (FAQ)
How do the A260 conversion factors work?
At a 10 mm equivalent path, conventional mass-concentration factors are about 50 ng/µL per A260 for dsDNA, 40 for RNA and 33 for ssDNA. These are empirical estimates. Base composition, structure and chemical modifications affect absorbance; use a sequence-specific extinction coefficient for precise oligo quantification.
Does a low A260/A230 ratio identify a contaminant?
No. Extraction reagents such as guanidine or phenol may contribute, but a blank mismatch or measurement noise can also change this ratio. Very high ratios can occur when A230 is close to zero. Review the instrument spectrum, blank and measurement range instead of assigning a contaminant from one ratio.
How do pH and the blank affect A260/A280?
pH and ionic strength affect absorbance ratios. Use a blank that matches the sample buffer and follow the instrument and extraction protocol. A ratio near 1.8 for DNA or 2.0 for RNA is an approximate reference, not a pH-independent proof of purity.
Can three absorbance readings reconstruct a spectrum or separate DNA from RNA?
No. The reference curves illustrate typical patterns; they are not reconstructed from your inputs. Three absorbance values cannot locate peaks or determine the complete spectrum. Standard A260 quantification includes DNA, RNA and other UV-absorbing compounds. Use an actual wavelength scan and a validated DNA- or RNA-selective assay when needed.
Share This Page
Recommend Tools
DNA Ligation Molar Ratio Calculator
BiologyCalculate optimal insert-to-vector molar ratios (3:1, 5:1, 1:1), picomole quantities, and generate bench-ready T4 DNA ligase master mix recipes
Primer Resuspension & Dilution Calculator
BiologyCalculate volume of water or TE buffer to reconstitute lyophilized primers to 100 µM stock and prepare 10 µM PCR working solutions
DNA Copy Number Calculator
BiologyConvert DNA mass and copies with dsDNA/ssDNA options, fragment length, and concentration output
Explore All Lab Calculators →