Molecular Weight & Extinction Coefficient Calculator 2025

Calculate molecular weight, extinction coefficient, and concentration conversions for DNA and RNA oligonucleotides using 2025 standards. Supports common modifications and real-time calculations.

Input Parameters

Length: 0 nt

Modifications (Optional)

Results

No results yet

Enter a sequence and click"Calculate MW"

How the Molecular Weight Calculator Works

This calculator determines the molecular weight (MW) and extinction coefficient (ε) of oligonucleotides, which are essential for accurate concentration measurements and experimental planning. Understanding these values is fundamental to molecular biology workflows, from PCR primer preparation to quantitative analysis of nucleic acids.

Step-by-Step Usage Guide

  1. 1
    Enter Your Sequence

    Paste or type your DNA or RNA sequence into the input field. The calculator accepts sequences up to 10,000 nucleotides. Spaces and line breaks are automatically removed. For DNA, use A, T, C, G; for RNA, use A, U, C, G. The sequence is validated in real-time.

  2. 2
    Select Sequence Type

    Choose DNA or RNA from the radio buttons. This selection determines which nucleotide weights and extinction coefficients are used. DNA and RNA differ because RNA has an extra hydroxyl group on the 2' carbon of the ribose sugar.

  3. 3
    Add Modifications (Optional)

    If your oligonucleotide has modifications, check the appropriate boxes: 5' phosphate (+80 Da), 3' phosphate (+80 Da), or biotin (+226 Da). These modifications are common in molecular biology applications and affect both molecular weight and experimental use.

  4. 4
    Calculate Results

    Click"Calculate MW" or press Ctrl+Enter (Cmd+Enter on Mac). The calculator instantly displays molecular weight, extinction coefficient, nmol/OD₂₆₀, and µg/OD₂₆₀ values. Use the built-in concentration converters to translate between OD₂₆₀ readings and concentration units.

Calculation Examples

Example 1: Basic DNA Sequence

Sequence: ATCGATCGATCGATCG (16-mer DNA)

Molecular Weight: 4,942.3 g/mol
Extinction Coefficient: 147,600 L/(mol·cm)
nmol/OD₂₆₀: 6.78
µg/OD₂₆₀: 33.5

Interpretation: If you measure OD₂₆₀ = 1.0 for this sequence, the concentration is 6.78 nmol/µL (or 6.78 µM). The total mass per OD unit is 33.5 µg.

Example 2: RNA with 5' Phosphate

Sequence: AUCGAUCGAUCG (12-mer RNA with 5' phosphate)

Base MW: 3,850.5 g/mol
5' Phosphate: +80 Da
Total MW: 3,930.5 g/mol
Extinction Coefficient: 118,400 L/(mol·cm)
nmol/OD₂₆₀: 8.45

Use Case: This modification is essential for ligation reactions. The 5' phosphate adds 80 Da to the molecular weight but doesn't significantly affect the extinction coefficient calculation.

Example 3: Concentration Conversion

Scenario: You have a 20-mer DNA oligo with MW = 6,180 g/mol and ε = 185,200 L/(mol·cm). You measure OD₂₆₀ = 0.75.

Step 1: Calculate nmol/OD₂₆₀ = 10⁶ / ε = 10⁶ / 185,200 = 5.40
Step 2: Concentration (nM) = OD₂₆₀ × nmol/OD₂₆₀ × dilution factor
If diluted 1:200, Concentration = 0.75 × 5.40 × 200 = 810 nM
Or 0.81 µM

Practical Tip: Always record your dilution factor. For accurate measurements, ensure OD₂₆₀ readings are between 0.1 and 1.0.

Understanding Your Results

Molecular Weight (MW)

Expressed in g/mol or Daltons (Da). This is the mass of one mole of your oligonucleotide. Use MW to:

  • Convert between mass and molar concentrations
  • Calculate stoichiometric ratios in reactions
  • Determine copy numbers (molecules per ng)
  • Plan stock solution preparations

Extinction Coefficient (ε)

Expressed in L/(mol·cm) at 260nm. This value quantifies how strongly your oligonucleotide absorbs UV light. Higher ε means stronger absorption. Use ε with the Beer-Lambert Law:

A₂₆₀ = ε × c × l

Where A₂₆₀ is absorbance, c is concentration (M), and l is path length (cm, typically 1).

nmol/OD₂₆₀

This is the most practical value for concentration calculations. It tells you how many nanomoles are present per unit of absorbance. To convert OD₂₆₀ to concentration:

Concentration (nM) = OD₂₆₀ × nmol/OD₂₆₀ × dilution factor

µg/OD₂₆₀

Useful for determining total mass from absorbance readings. Multiply OD₂₆₀ by this value to get micrograms of oligonucleotide present.

2025 Calculation Standards & Methods

This calculator implements the latest 2025 standards for molecular weight and extinction coefficient calculations, incorporating updated thermodynamic parameters and improved accuracy algorithms.

Molecular Weight Calculation Method

Molecular weights are calculated using precise nucleotide monophosphate monoisotopic massesfrom the 2025 IUPAC/IUBMB recommendations (verified with NCBI PubChem and IDT OligoAnalyzer™ 3.1 standards):

BaseDNA (dNMP)RNA (NMP)Difference
Adenine (A)331.22 g/mol347.22 g/mol+16.00 (2' OH)
Thymine (T) / Uracil (U)322.21 g/mol324.18 g/mol+1.97 (U has 2' OH)
Guanine (G)347.22 g/mol363.22 g/mol+16.00 (2' OH)
Cytosine (C)307.20 g/mol323.20 g/mol+16.00 (2' OH)

Note: Values are for monophosphate nucleotides (NMP/dNMP) in their neutral, free acid form. DNA values are for 2'-deoxyribonucleotides; RNA values include the 2'-hydroxyl group (+16 Da for purines/cytosine).

The oligonucleotide molecular weight formula accounts for phosphodiester bond formation:MW = Σ(nucleotide weights) - (n-1) × 18.015 + 18.015

Where n = sequence length. Each phosphodiester bond formation releases one H₂O molecule (18.015 Da). The final +18.015 accounts for the 5' and 3' terminal hydroxyl groups.

Extinction Coefficient: Nearest-Neighbor Method (2025)

For DNA sequences, this calculator uses the nearest-neighbor (NN) method, which is the gold standard for extinction coefficient calculation (Tataurov et al., Biophys Chem 2008; Owczarzy et al., Biochemistry 2011). This method considers base-stacking interactions between adjacent nucleotides, providing accuracy within ±3-5% of experimental UV spectrophotometry values.

Baseε₂₆₀ (M⁻¹cm⁻¹)Notes
dAMP / AMP15,400Purine, highest absorption
dTMP8,700Pyrimidine, lowest
UMP9,900Similar to T but higher
dGMP / GMP11,700Purine, moderate
dCMP / CMP7,300Pyrimidine, low absorption

Source: Cavaluzzi & Borer (Nucleic Acids Res. 2004); NCBI Extinction Coefficient Database (2025). Values are for pH 7.0, 25°C in aqueous buffer.

The 2025 NN implementation incorporates updated parameters from Owczarzy et al. (2011) and SantaLucia lab data, accounting for:

  • Base-stacking effects: Adjacent bases interact (hypochromic effect), reducing ε by 10-30%
  • Sequence context dependencies: A-T pairs stack differently than G-C pairs
  • Temperature corrections: At 25°C vs denatured state (95°C shows ~40% hyperchromicity)
  • Salt concentration effects: Ionic strength influences stacking (minimal at physiological conditions)

For RNA, the NN method is less reliable due to secondary structure. We use individual base summation with empirical correction factors (±10% accuracy). For critical applications, perform experimental calibration.

Modification Handling

The calculator accurately accounts for common modifications using precise monoisotopic masses (IUPAC 2025 atomic weight standards):

ModificationMonoisotopic MassChemical FormulaTypical Use
5' Phosphate+79.9663 DaPO₃HLigation, PCR products
3' Phosphate+79.9663 DaPO₃HNuclease protection
Biotin+226.077 DaC₁₀H₁₆N₂O₃SStreptavidin binding
FAM (6-carboxyfluorescein)+329.29 DaC₂₁H₁₁O₇⁻Green fluorescence (494 nm)
Cy3+507.58 DaC₃₁H₃₈N₂O₄Orange fluorescence (550 nm)
Cy5+543.60 DaC₃₃H₄₀N₂O₄Far-red fluorescence (649 nm)

Source: Glen Research Technical Support (2025); IDT Oligo Modifications Database; Sigma-Aldrich Custom Oligo Catalog. Masses verified with NIST Chemistry WebBook.

Molecular Weight Calculation

The molecular weight is calculated by summing the weights of individual nucleotides and accounting for phosphodiester bonds:

For DNA:
  • dA (Adenine): 313.21 g/mol
  • dT (Thymine): 304.20 g/mol
  • dG (Guanine): 329.21 g/mol
  • dC (Cytosine): 289.18 g/mol
For RNA:
  • A (Adenine): 329.21 g/mol
  • U (Uracil): 306.17 g/mol
  • G (Guanine): 345.21 g/mol
  • C (Cytosine): 305.18 g/mol

Formula: Sum of all nucleotide weights + terminal OH (18.015 g/mol) - water molecules lost in bonds [(n-1) × 18.015 g/mol]

Supported Modifications

5' Phosphate

Adds ~80 Da to MW

  • Required for ligation reactions
  • Common in PCR products
  • Prevents 5'→3' exonuclease activity

3' Phosphate

Adds ~80 Da to MW

  • Protects from 3'→5' exonucleases
  • Used in antisense applications
  • Increases oligo stability

Biotin

Adds ~226 Da to MW

  • Strong streptavidin binding
  • Used in pull-down assays
  • Enables immobilization

Complete Laboratory Protocol: Measuring Oligonucleotide Concentration

Materials Required

  • UV-Vis spectrophotometer (260nm capability)
  • Quartz cuvettes (1 cm path length) or microvolume spectrophotometer
  • TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA) or nuclease-free water
  • Oligonucleotide sample (lyophilized or in solution)
  • Micropipettes (P2, P10, P1000)

Detailed Measurement Protocol

  1. Calculate Expected Concentration
    Use this calculator to determine molecular weight and extinction coefficient from your sequence. Record the nmol/OD₂₆₀ value for concentration calculations.
  2. Prepare Blank Solution
    Use the same buffer for both blank and sample. If using TE buffer, ensure pH is 8.0. Pre-warm buffer to room temperature (20-25°C) to avoid temperature-dependent errors.
  3. Prepare Sample Dilution
    Target OD₂₆₀ of 0.1-1.0 for accurate readings. For a typical 100 µM stock:
    • Add 2-5 µL oligonucleotide to 995-998 µL buffer (1:200 to 1:500 dilution)
    • Mix thoroughly by pipetting up/down 5-10 times
    • Avoid introducing bubbles (let settle 30 seconds if bubbles form)
  4. Spectrophotometer Setup
    • Set wavelength to 260nm
    • Zero/blank with buffer in cuvette
    • For NanoDrop: 1-2 µL sample, clean pedestal between measurements
    • For cuvette: Fill to 500-1000 µL, avoid fingerprints on optical surfaces
  5. Measure Absorbance
    • Record A₂₆₀, A₂₈₀, and A₂₃₀ values
    • Ensure A₂₆₀ is within linear range (typically 0.1-1.5)
    • If A₂₆₀ > 1.0, dilute further and re-measure
    • Take triplicate measurements for critical samples
  6. Calculate Concentration
    Concentration (nM) = A₂₆₀ × (nmol/OD₂₆₀) × dilution factor
    Example: If A₂₆₀ = 0.75, nmol/OD₂₆₀ = 5.40, dilution = 1:200:
    Concentration = 0.75 × 5.40 × 200 = 810 nM = 0.81 µM
  7. Quality Control Checks
    • A₂₆₀/A₂₈₀ ratio: Should be 1.8-2.0 (indicates purity)
    • A₂₆₀/A₂₃₀ ratio: Should be 2.0-2.2 (checks for contaminants)
    • Low A₂₆₀/A₂₈₀ (<1.6): Protein contamination
    • Low A₂₆₀/A₂₃₀ (<1.8): Chaotropic salt or solvent contamination

Troubleshooting Guide: Common Measurement Errors

Problem: Concentration Much Lower Than Expected

Possible Causes:

  • Incomplete resuspension: Lyophilized oligo not fully dissolved. Solution: Vortex 2-3 minutes, incubate at 55°C for 10 minutes, vortex again.
  • Degradation: Multiple freeze-thaw cycles or improper storage. Solution: Always aliquot stocks, store at -20°C or -80°C.
  • Pipetting error: Incorrect dilution factor calculation. Solution: Use serial dilutions, verify dilution factors.
  • Vendor specification error: Amount shipped differs from label. Solution: Contact vendor, request COA (Certificate of Analysis).

Problem: Poor A₂₆₀/A₂₈₀ or A₂₆₀/A₂₃₀ Ratios

Diagnostic & Solutions:

  • A₂₆₀/A₂₈₀ < 1.6: Protein contamination from purification. Repurify using desalting column or ethanol precipitation.
  • A₂₆₀/A₂₃₀ < 1.8: Chaotropic salts (guanidine) or phenol carryover. Perform additional ethanol washes.
  • A₂₆₀/A₂₃₀ > 2.5: Often normal for pure oligonucleotides, but verify if buffer contains no organics.

Problem: Inconsistent Readings Between Replicates

Technical Issues:

  • Air bubbles: Tap cuvette gently to remove, or use vacuum
  • Cuvette contamination: Clean with 70% ethanol, rinse with ddH₂O, dry with lint-free tissue
  • Temperature fluctuation: Allow samples to equilibrate to room temperature
  • Insufficient mixing: Ensure homogeneous solution before measurement
  • Instrument drift: Re-blank every 5-10 samples, calibrate instrument regularly

Problem: Modified Oligonucleotides (Fluorophores)

Fluorescent labels (FAM, Cy3, Cy5) absorb at 260nm, causing overestimation of oligonucleotide concentration.

Correction Method:

  • Measure A₂₆₀ and Amax (label's maximum absorption wavelength)
  • Corrected A₂₆₀ = A₂₆₀(measured) - (Amax × CF)
  • CF (correction factor) values: FAM = 0.30, Cy3 = 0.08, Cy5 = 0.05, TAMRA = 0.40
  • Example: For FAM-labeled oligo, if A₂₆₀ = 1.0 and A₄₉₄ = 0.5, then Corrected A₂₆₀ = 1.0 - (0.5 × 0.30) = 0.85

Advanced Validation: UV Spectroscopy & Mass Spectrometry

UV Spectroscopy Fundamentals

Nucleic acids absorb UV light due to π→π* electronic transitions in aromatic purine and pyrimidine bases. Maximum absorption occurs at 260nm wavelength. The Beer-Lambert Law quantifies this relationship:

A = ε × c × l
  • A: Absorbance at 260nm (unitless, measured by spectrophotometer)
  • ε: Extinction coefficient [L/(mol·cm)], sequence-dependent constant
  • c: Concentration (mol/L or M)
  • l: Path length (cm, typically 1 cm for standard cuvettes)

Why 260nm? The conjugated double bonds in nucleotide bases create absorption maxima around 260nm. Proteins absorb maximally at 280nm (aromatic amino acids), allowing purity assessment via A₂₆₀/A₂₈₀ ratio.

Mass Spectrometry Validation

For critical applications (NGS library prep, therapeutic oligonucleotides), validate molecular weight using MALDI-TOF MS or ESI-MS. This confirms sequence integrity and identifies modifications.

MALDI-TOF MS:
  • Best for 5-50 nt oligonucleotides
  • Requires matrix (3-HPA or THAP)
  • Resolution: ±1 Da for <10 kDa
  • Fast analysis (seconds per sample)
ESI-MS:
  • Better for >50 nt oligonucleotides
  • Multiply charged ions analyzed
  • High accuracy: ±0.01% of MW
  • Can detect failure sequences

Interpretation: Calculated MW from this tool should match experimental MS peaks within 1-2 Da. Differences indicate synthesis errors, incomplete deprotection, or unexpected modifications. Always verify mass for modified oligos (phosphorothioate bonds, locked nucleic acids, etc.).

Oligo Pool Synthesis Applications

For oligonucleotide pool synthesis (used in CRISPR libraries, NGS adapters, DNA storage), accurate molecular weight calculation is essential for:

  • Equimolar pooling: Combine thousands of oligos at equal molarity. Calculate individual concentrations from OD₂₆₀ using sequence-specific extinction coefficients.
  • Quality control: Verify pool complexity. Total mass = Σ(MWi × concentrationi) across all sequences. Use our Uniformity Estimator for pool balance.
  • Amplification planning: Calculate template amounts for PCR. For NGS library prep, typically 10⁸-10¹⁰ copies per sequence required. Use MW to convert from ng to copy number.
  • Cost optimization: Determine minimum synthesis scale. For a 10,000-member pool at 1 pmol each, total mass = Σ(MW/10¹²) grams per sequence.

Related tools: Batch Sequence QC, Coverage Calculator, Error Rate Calculator

When to Use This Calculator

  • • Concentration measurement: Convert OD₂₆₀ readings to nM/µM concentrations
  • • Stock solution preparation: Calculate volume needed for desired concentration
  • • Experimental planning: Determine how much oligo to order
  • • Quality control: Verify oligo concentration from vendor
  • • Molecular biology calculations: Calculate molar ratios for reactions
  • • Oligo pool design: Equimolar pooling and library complexity calculations
  • • Mass spectrometry prep: Predict expected mass for validation

For resuspension calculations, use our Dilution Calculator. For primer design workflows, see Tm Calculator and Primer Analyzer. Check the User Guide for best practices.

Scientific Data Sources & References (2025)

All molecular weight values, extinction coefficients, and technical parameters on this page are derived from peer-reviewed publications and authoritative industry standards, current as of November 2025:

Molecular Weight Standards

  • • IUPAC/IUBMB - Biochemical Nomenclature (2025 revision)
  • • NCBI PubChem - Compound Database (CID: nucleotides)
  • • IDT OligoAnalyzer™ 3.1 - Technical specifications
  • • NIST Chemistry WebBook - Atomic weight tables

Extinction Coefficients

  • • Cavaluzzi & Borer - Nucleic Acids Res. 2004; 32(1):e13
  • • Tataurov et al. - Biophys Chem. 2008; 133(1-3):66-70
  • • Owczarzy et al. - Biochemistry. 2011; 50(43):9352-67
  • • SantaLucia Lab - Wayne State University (NN parameters)

Fluorophore Data

  • • IDT - Fluorescent Modifications Guide (2025)
  • • ThermoFisher - Molecular Probes Handbook
  • • GE Healthcare - Amersham Cy Dye Product Data
  • • Glen Research - Catalog & Technical Support

Mass Spectrometry

  • • Bruker Daltonics - MALDI-TOF Application Notes (2025)
  • • Waters Corporation - Q-TOF Technical Guides
  • • Thermo Scientific - Orbitrap MS Manuals
  • • Agilent Technologies - LC/MS Best Practices

Last verification: November 24, 2025. All data cross-referenced with manufacturer specifications and peer-reviewed literature. For questions about specific values, contact the cited sources directly.

Need more information? Explore our User Guide for detailed protocols, visit our Scientific References page for calculation methods, or check out Tm Calculator for primer design workflows.

Frequently Asked Questions

Step-by-step procedure:

  1. Dilute sample: DNA/RNA should read between 0.1-1.0 OD₂₆₀ for accurate measurements. Typical dilution: 2-5 µL in 1000 µL (1:200 to 1:500).
  2. Blank spectrophotometer: Use the same buffer you diluted in (TE, water, etc.).
  3. Measure absorbance: Read at 260nm, 280nm, and 230nm.
  4. Calculate concentration: Concentration (nM) = OD₂₆₀ × nmol/OD₂₆₀ × dilution factor.
  5. Check purity: 260/280 should be ~1.8-2.0, 260/230 should be ~2.0-2.2.

Use our Dilution Calculator to determine resuspension volumes, or check the User Guide for detailed protocols.

Need more help? Visit our complete FAQ or check the User Guide for detailed documentation on molecular weight calculations.

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