OligoEvaluator™ for T_m Calculation & Primer Analysis

OligoEvaluator™ for DNA or RNA Primers

We are pleased to offer OligoEvaluator™, our online oligonucleotide sequence calculator that provides primer analysis values for PCR:

• Base count
• Molecular weight
• Extinction coefficient
• Oligo type
• µg/OD at 260 nm
• Length (base pairs)
• Primer melting temperature T_m (°C)
• GC content %
• GC clamp
• Run length (bp)
• Secondary structure
• Primer dimer check
  
• BLAST sequence link

OligoEvaluator™ is easy to use: select DNA or RNA, paste your sequence, and click calculate for the OligoEvaluator™ tool to return values. All reported properties are available for export to a convenient Excel template.

Compute Oligo Properties, Resuspension & Dilution

The OligoEvaluator™ consists of three modules. Choose analysis to look at the complete properties of up to 10 primers at a time. Resuspension and Dilution are separate calculator modules within OligoEvaluator™.

Modules	Features
Analysis	Enter up to 10 sequences at a time, and the tool returns values for all major physical properties, such as molecular weight, melting temperature, secondary structure, and primer dimer formation (secondary structure and primer dimer formation information provided in simple-to-interpret text format, e.g. secondary structure--strong)
Resuspension	Enter the quantity, desired concentration, and molecular weight, and the tool returns the volume of water or buffer needed to resuspend a dry oligonucleotide
Dilution	Enter the stock (resuspension) concentration, desired final volume, and desired final concentration, and the tool returns the volume of stock solution needed for dilution

Calculator Advanced Features
Feature	Description
Reaction Conditions	Review secondary structure analysis and primer dimer formation to check for problematic folding
BLAST Search	Check for oligonucleotide sequence homology

Reaction Conditions

Definitions and values used in the OligoEvaluator™ calculator

Parameter	Value
Oligo Concentration	The default value = 250.0 nM.
Monovalent Ion Concentration	This is the concentration of all monovalent ions, e.g. K+, present in the reaction mixture. K+ encourages annealing by reducing charge repulsion between the negatively charged primers and template backbones. The default value = 100.0 mM.
Free Mg++ Ion Concentration	This is the concentration of Mg2+ (magnesium ion) in the reaction mixture. Mg2+ is a cofactor required by thermostable DNA polymerases. The default value = 5.0 mM.
Total Na[+] Equivalent	The sodium equivalent is calculated as follows: [Na+] = Monovalent ion concentration + 4 × (Free Mg2+) 1/2 (all in molarity) The default value = 382.84 mM
Temperature for Free Energy Calculation	This is used to calculate the Gibbs free energy (ΔG) in the formula: ΔG = ΔH - T ΔS. The default value = 25.0 °C (298K).

How do you calculate the annealing temperature of a primer?

The OligoEvaluator™ provides melting temperature for oligos. Just enter you sequence in the calculator Analysis module, and you'll find the T_m reported in the seventh column. Annealing temperature should usually be several degrees below the T_m value. You can find more detailed information about melting temperature in our article about Oligonucleotide Melting Temperature.