Oligonucleotide Properties Calculator

The calculator provides three Tm calculation methods, each with different accuracy levels:

• Basic Tm: Uses a simple formula (Tm = 4(G+C) + 2(A+T) for short sequences, or Tm = 64.9 + 41×(G+C-16.4)/length for longer sequences). This method provides a quick estimate but is less accurate, especially for sequences outside the 14-20 nucleotide range. It assumes standard conditions (50 nM primer, 50 mM Na⁺, pH 7.0) and does not account for salt variations.
• Salt-Adjusted Tm: Applies salt concentration correction to the basic Tm formula. For sequences <14 nt: Tm = (A+T)×2 + (G+C)×4 - 16.6×log₁₀(0.050) + 16.6×log₁₀([Na⁺]). For sequences ≥14 nt: Tm = 100.5 + 41×(G+C)/length - 820/length + 16.6×log₁₀([Na⁺]). This method accounts for ionic strength effects, as sodium ions stabilize double-stranded DNA through charge neutralization. However, it still lacks the precision of nearest-neighbor methods.
• Nearest-Neighbor Tm: Uses the SantaLucia 1998 nearest-neighbor thermodynamic method, which is the industry standard and most accurate for sequences 8-40 nucleotides long. This method calculates ΔH (enthalpy) and ΔS (entropy) by summing nearest-neighbor pair contributions, accounting for base stacking interactions. It solves: Tm = ΔH / (ΔS + R × ln([primer]/4)). This is the recommended method for PCR primer design and provides accuracy within 1-2°C.

Recommendation: For PCR primer design, always use the nearest-neighbor Tm. Use basic or salt-adjusted methods only for quick estimates or when comparing with older literature values.

To calculate concentration from OD260 readings, enter your measured absorbance value in the"Measured Absorbance at 260 nm" field. The calculator will automatically calculate:

• Concentration (µM): Calculated as C = OD₂₆₀ / (ε × pathlength), where ε is the extinction coefficient and pathlength is assumed to be 1 cm. The result is displayed in micromolar (µM) for convenience.
• Micrograms: Calculated as Mass = OD₂₆₀ × µg/OD₂₆₀. This tells you the total mass of oligonucleotide in your sample.

Example: If you measure OD₂₆₀ = 1.0 for a 20-mer DNA primer, and the calculator shows nmol/OD₂₆₀ = 5.0, then:

• Concentration = 1.0 × 5.0 = 5.0 µM
• If the solution volume is 1 ml, total amount = 5.0 nmol or ~25 µg

Note: If you don't have a measured absorbance, you can still see the extinction coefficient and conversion factors (nmol/OD₂₆₀ and µg/OD₂₆₀) for future use. These values are sequence-specific and remain constant for a given oligonucleotide.

Thermodynamic constants describe the stability and energetics of DNA/RNA hybridization. All values are calculated for standard conditions: 1 M NaCl at 25°C at pH 7.0.

• ΔH (deltaH, Enthalpy): Measured in kcal/mol, represents heat released or absorbed during hybridization. More negative values indicate more stable duplexes because more heat is released when strands bind. Typical values range from -50 to -150 kcal/mol for oligonucleotides.
• ΔS (deltaS, Entropy): Measured in cal/(mol·K), represents the change in disorder during hybridization. Negative values indicate increased order (less random) when single strands form a double helix. Typical values range from -100 to -300 cal/(mol·K).
• ΔG (deltaG, Free Energy): Measured in kcal/mol, calculated as ΔG = ΔH - T × ΔS, where T is temperature in Kelvin. This is the overall stability indicator—more negative ΔG values indicate more stable structures. At the melting temperature, ΔG = 0 (equilibrium between bound and unbound states). Typical values at 25°C range from -5 to -30 kcal/mol.
• RlnK: Measured in cal/(mol·K), related to the equilibrium constant K. Calculated as RlnK = -ΔG / T. This value relates to the ratio of bound to unbound strands at equilibrium. Higher positive values indicate more stable hybridization.

Practical Applications:

• Use ΔG to compare stability of different sequences or structures
• Predict hybridization behavior at different temperatures
• Design experiments with optimal annealing temperatures
• Understand why some sequences form stable duplexes while others don't

These constants are calculated using nearest-neighbor parameters from Sugimoto et al. (1996) and SantaLucia (1998), which account for base stacking energies and terminal effects.

The simplified hairpin/self-dimer calculations in this calculator provide basic thresholds (minimum base pairs required) but lack the detailed analysis needed for accurate predictions. These calculations are based on homology and length constraints but don't account for:

• Loop size and stability
• Base stacking energies in stems
• Temperature-dependent effects
• Detailed ΔG calculations for each structure
• Visualization of predicted structures

For comprehensive secondary structure analysis, including detailed hairpin predictions, self-dimer detection, hetero-dimer analysis, and structure visualization, use the dedicated Secondary Structure Predictor tool. This tool uses advanced algorithms based on nearest-neighbor thermodynamics and provides:

• Detailed ΔG calculations for each structure
• Risk level assessment (low/medium/high)
• Structure visualization
• Position information for problematic regions
• Recommendations for sequence modifications

The deprecated calculations are still shown for comparison purposes but should not be relied upon for critical primer design decisions.

Yes! Select"RNA" as the sequence type. The calculator uses RNA-specific calculations throughout:

• Sequence Input: Use A, U, C, G (U replaces T in RNA)
• Molecular Weight: RNA nucleotides have different weights due to the 2'-OH group. Formula: MW = Σ(nucleotide weights) - (n-1) × 18.015 + terminal corrections. Standard weights: A=329.21, U=306.17, C=305.18, G=345.21 g/mol. Additional 159 g/mol for 5'-triphosphate if present.
• Extinction Coefficient: Uses RNA-specific base extinction coefficients from Cavaluzzi & Borer (2004): A=15,400, U=9,900, C=7,300, G=11,700 L/(mol·cm) at pH 7.0. These revised values provide improved accuracy over older literature values.
• Tm Calculation: Uses RNA-specific empirical formula: Tm = 79.8 + 18.5×log₁₀([Na⁺]) + 58.4×(G+C)/length + 11.8×((G+C)/length)² - 820/length (Freier et al., 1986). RNA typically has 10-15°C higher Tm than equivalent DNA sequences due to A-form helix geometry and 2'-OH interactions.
• Reverse Complement: Correctly handles U→A complementarity (instead of T→A for DNA).

Note: RNA thermodynamic parameters differ from DNA due to structural differences (A-form vs. B-form helix). The calculator accounts for these differences automatically when RNA is selected.

The calculator uses established scientific methods with the following accuracy levels:

• Molecular Weight: Accurate within 0.1-0.5% for standard sequences. Accuracy decreases slightly with modifications or non-standard bases.
• Extinction Coefficient: Nearest-neighbor method provides accuracy within 2-5% for DNA sequences. Simple sum method for RNA has similar accuracy.
• Nearest-Neighbor Tm: Most accurate method, typically within 1-2°C for sequences 8-40 nucleotides long. Accuracy decreases for sequences outside this range or with modifications.
• Basic/Salt-Adjusted Tm: Less accurate, typically within 3-5°C. Useful for quick estimates but not recommended for critical applications.
• Thermodynamic Constants: Based on published nearest-neighbor parameters, accurate for standard conditions (1 M NaCl, 25°C, pH 7.0).

Factors that affect accuracy:

• Sequence Length: Nearest-neighbor parameters optimized for 14-20mers. Accuracy decreases for shorter (<8 nt) or longer (>40 nt) sequences.
• Salt Concentration: Calculations assume monovalent cations (Na⁺, K⁺). Divalent cations (Mg²⁺, Mn²⁺) significantly affect Tm but are not accounted for.
• pH: All calculations assume pH 7.0. Changes in pH affect base pairing stability.
• Modifications: Chemical modifications (fluorophores, biotin, etc.) may affect properties but are not fully accounted for in calculations.
• Solvents/Additives: Presence of formamide, DMSO, detergents, or other additives affects actual melting temperatures but are not included in calculations.
• Sequence Composition: Extreme GC content (<20% or >80%) or homopolymer runs may reduce accuracy.

Recommendation: For experimental applications, always validate critical parameters empirically. Use calculated values as starting points for experimental design, then optimize based on actual results.

Important assumptions and limitations:

• Salt Concentration: All Tm calculations assume monovalent cations (Na⁺ or K⁺) between 0.01 and 1.0 M. Divalent cations (Mg²⁺, Mn²⁺) are not accounted for, although they significantly affect duplex formation (Nakano et al., 1999).
• Sequence Requirements: Nearest-neighbor calculations assume sequences are not symmetric, contain at least one G or C, and are at least 8 nucleotides long. Accuracy decreases for sequences longer than 40 nucleotides. Parameters were optimized for 14-20mers but remain reliable up to 40 nt.
• pH and Conditions: All calculations assume pH 7.0. Changes in pH, presence of detergents, solvents (ethanol, formamide), or other additives will affect actual melting temperatures.
• OD Calculations: Extinction coefficients assume single-stranded DNA/RNA. For double-stranded DNA, multiply ε by 0.92 to account for hypochromicity. Pathlength is assumed to be 1 cm.
• Molecular Weight: DNA calculations assume 5' and 3' hydroxyl groups (no phosphate). RNA calculations assume 5' triphosphate. Modifications may not be fully accounted for.
• Temperature: Thermodynamic constants are calculated at 25°C. Actual behavior may vary at different temperatures.

For sequences with modifications, extreme compositions, or non-standard conditions, consider using specialized tools or validating results experimentally.

This all-in-one calculator combines functionality from multiple specialized tools, making it ideal for comprehensive oligonucleotide characterization:

• vs. IDT OligoAnalyzer: Provides similar nearest-neighbor Tm calculations but with transparent methodology and free access. Includes all properties in one interface without requiring multiple tabs.
• vs. NEB Tm Calculator: Offers three Tm calculation methods (basic, salt-adjusted, nearest-neighbor) for comparison, plus additional properties like extinction coefficient and thermodynamic constants.
• vs. Thermo Fisher Calculator: Uses SantaLucia 1998 parameters (industry standard) and provides detailed explanations of calculation methods with scientific references.
• Unique Features: Combines Tm, molecular weight, extinction coefficient, OD260 conversion, reverse complement, and thermodynamic constants in a single calculation. Includes detailed method comparison and educational content.

When to use specialized tools: For batch processing, use our Batch Sequence QC. For detailed secondary structure analysis, use Secondary Structure Predictor. For primer pair analysis, use Primer Analyzer.

Recommended PCR Primer Design Workflow:

1. Initial Design: Design primers (typically 18-22 nt) with target GC content 40-60%. Aim for Tm between 55-65°C.
2. Calculate Properties: Enter primer sequence here. Verify length, GC content, and molecular weight. Note the nearest-neighbor Tm value.
3. Check Tm Match: For primer pairs, ensure forward and reverse primers have Tm within 5°C of each other. The nearest-neighbor method is most accurate for this comparison.
4. Verify Stability: Check thermodynamic constants. ΔG should be negative (around -10 to -30 kcal/mol for stable primers). More negative ΔG indicates stronger binding.
5. Secondary Structure Check: Use Secondary Structure Predictor to check for hairpins and self-dimers. Look for structures with ΔG more negative than -3 kcal/mol, which may cause problems.
6. Final Validation: Use Primer Analyzer for comprehensive primer pair analysis including cross-dimers and specificity.
7. Order Preparation: Note extinction coefficient and nmol/OD₂₆₀ for later quantification after synthesis. Use Dilution Calculator to prepare working stocks.

Pro Tip: For PCR annealing temperature, use Tm - 5°C as a starting point. Optimize empirically based on your specific conditions and polymerase.

Step-by-Step Solution Preparation:

1. Calculate Stock Concentration: Enter your sequence and measured OD₂₆₀ value. The calculator displays concentration in µM and total mass in µg.
2. Determine Target Concentration: Common working concentrations: 10 µM for PCR primers, 100 µM for long-term storage, 1 µM for qPCR.
3. Calculate Dilution: Use our Dilution Calculator to determine volumes. Formula: C₁V₁ = C₂V₂.
4. Example: If OD₂₆₀ = 10.0 gives 50 µM in 500 µL (25 nmol), to make 10 µM working stock: dilute 1:5 (100 µL stock + 400 µL buffer).
5. Storage: Store stock solutions at -20°C in small aliquots. Avoid repeated freeze-thaw cycles. Working stocks (10 µM) can be kept at 4°C for several weeks.

Quality Control: After dilution, verify concentration by measuring OD₂₆₀ of the diluted sample. Expected OD₂₆₀ = (concentration in µM) × (ε / 10⁶).