PCR Primer Tm Calculator
From Base Composition
Calculate Tm from the number of each base in the primer
Quick Tm (Wallace Rule)
Fast estimate for short primers using the 2+4 rule
Formula
Short primers (< 14 nt): Tm = 2(A+T) + 4(G+C). Longer primers: Tm = 64.9 + 41×(nGC − 16.4)/N. Salt-adjusted: Tm = Tm_basic + 16.6 × log₁₀([Na+]).
Frequently Asked Questions
What is primer Tm?
Primer Tm (melting temperature) is the temperature at which 50% of the primer molecules are bound to their complementary strand. It is critical for setting PCR annealing temperatures.
What is the Wallace rule?
The Wallace rule estimates Tm for short primers (< 14 nt): Tm = 2(A+T) + 4(G+C). For longer primers, the formula Tm = 64.9 + 41×(G+C−16.4)/N is more accurate.
How does salt concentration affect Tm?
Higher salt (Na+) concentration stabilizes DNA duplexes and increases Tm. The salt-adjusted formula adds 16.6 × log₁₀([Na+]) to the basic Tm.
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