What Is the DNA Melting Temperature (Tm) Calculator?
The melting temperature (Tm) is the temperature at which 50% of oligonucleotide duplexes are dissociated into single strands.
Accurate Tm prediction ensures specific primer-template hybridization while minimizing non-specific amplification and primer-dimer artifacts.
How Does the DNA Melting Temperature (Tm) Calculator Work?
The calculator strips non-alphabetical characters and tallies adenine (A), thymine (T), guanine (G), and cytosine (C) bases.
For oligonucleotides shorter than 14 bp, the Wallace rule Tm = 2(A+T) + 4(G+C) is applied.
For sequences ≥ 14 bp, the salt-corrected Marmur-Doty formula incorporates monovalent salt stabilization.
DNA Melting Temperature (Tm) Calculator Formula & Variables
The core mathematical equation utilized by this calculator is expressed as:
For primers ≥ 14 bp, salt-adjusted melting temperature accounts for base composition, cation concentration, and sequence length.
How to Use the DNA Melting Temperature (Tm) Calculator
- Paste your primer sequence in the 5′ to 3′ orientation.
- Set your PCR buffer monovalent salt concentration (default is 50 mM).
- Read Tm, %GC content, and recommended thermal cycling annealing temperature Ta.
Step-by-Step Example Calculation
18-mer primer with 50% GC
Input Values:
Understanding Your Result
Ideal PCR primers have Tm between 52°C and 65°C and 40%–60% GC content.
Forward and reverse primers should ideally match within 2°C to 3°C of each other.
Factors That Affect the Result
- GC Content: Triple hydrogen bonds in G-C pairs provide higher thermodynamic stability than double-bonded A-T pairs.
- Salt Concentration: Cations shield repulsive electrostatic charges along negatively charged phosphate backbones.
When Should You Use This Calculator?
- Designing PCR primers for colony PCR, qPCR, mutagenesis, or Gibson assembly.
- Sizing hybridization probes for Southern/Northern blots and microarray assays.
Assumptions & Limitations
- Does not model secondary hairpins or mismatched wobble bases.
Frequently Asked Questions
Calculation Accuracy & Reference Note
Implements standard salt-adjusted Marmur-Schildkraut empirical formulation.