PCR Annealing Temperature Calculator
From Primer Tm Values
Calculate annealing temperature from forward and reverse primer Tm
From Primer Composition
Estimate annealing temperature from primer length and GC content
Formula
Ta = Lowest Tm − 5 °C. Alternative: Ta = (Tm_forward + Tm_reverse) / 2 − 5 °C. Touchdown PCR: start at higher Tm, decrease by 1°C per cycle to (lowest Tm − 5°C).
Frequently Asked Questions
How do you determine annealing temperature?
The standard guideline is to set the annealing temperature 5°C below the lowest Tm of your primer pair. This ensures both primers bind efficiently while minimizing non-specific binding.
What is touchdown PCR?
Touchdown PCR starts with a high annealing temperature (near the higher Tm) and gradually decreases it over successive cycles. This increases specificity in early cycles and ensures efficient amplification in later cycles.
What if my primers have very different Tm values?
If the Tm difference exceeds 5°C, consider redesigning one or both primers to bring them closer together. Large Tm differences can lead to inefficient PCR with non-specific products.
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