Formula to Calculate the Annealing Temperature of Oligonucleotides for PCR
Last modified on July 24, 2026 • 1 min read • 91 wordsOligonucleotide annealing temperature rule of thumb
The thumbrule for calculating the annealing temperature for a PCR primer isTm (°C) = 81.5 + 0.41(%GC) - (675/N) where %GC is the percentage of G and C nucleotides in the oligo and N is the length of the oligo given in nucleotides.
Example calculation
5’-TTAAAATGATAACCATCTCGC-3' Tm = 81.5 + 0.41(33.3) - (675/21) Tm = 81.5 + 13.7 - 32.1 (rounded to one decimal) Tm = 63.1°C
When choosing the temperature for the PCR annealing step a good starting point is Tm - 5°C, for the above example that is 58°C.