Enter your Primer #1 and Primer #2 sequences (5' to 3'), choose a calculation method, and dial in your primer, salt, and Mg²⁺ concentrations to get a recommended annealing temperature — plus individual Tm values and GC content for each primer.
Recommended Annealing Temperature
--
Primer #1 Tm
--
Primer #2 Tm
--
Primer #1 GC Content
--
Primer #2 GC Content
--
The basic method uses the Wallace rule (4°C for each GC, 2°C for each AT), while the nearest neighbor method considers the thermodynamic properties of adjacent base pairs for more accurate results, especially for primers longer than 14 nucleotides.
The optimal annealing temperature is typically 3-5°C below the lowest primer Tm. This calculator automatically determines the recommended temperature based on your primer sequences and reaction conditions.
Higher salt concentrations increase Tm by stabilizing the DNA duplex. Typical PCR buffers contain 50-200 mM Na+ and 1.5-3.0 mM Mg²⁺. Accurate concentrations are important for precise Tm calculations.
Most PCR reactions use 0.2-0.8 µM of each primer. Higher concentrations can lead to primer-dimer formation, while lower concentrations may reduce PCR efficiency.
GC content affects primer stability and Tm. Ideal primers have 40-60% GC content. Very high GC content (>70%) can cause secondary structures, while very low GC content (<30%) may result in weak primer binding.
This calculator assumes perfect complementarity between primer and template. For primers with intentional mismatches (e.g., for mutagenesis), calculate Tm only for the matching portion of the primer sequence.
If primer Tm values differ by more than 5°C, consider redesigning one primer to match better. Large Tm differences can lead to unequal amplification efficiency and poor PCR performance.