The melting temperature of a specific oligonucleotide primer (T 
)
can be calculated by several different ways. The used primer pairs should be
constructed to have a similar T . The
simplest equation, often used, is
where A, C, G, and T symbolize the count of the particular nucleotide in the
primer. This formula was developed for hybridization assays with
oligonucleotides at a salt concentration of 1 M [93]. The equation is
inaccurate for calculation of T of primers longer than 20 nt.
Usually, the chosen annealing temperature is about 5psy176 C below the
calculated T and is determined by trial
and error.
Other equations used are
where 
is the concentration of monovalenced cations and l is
the length of the oligonucleotide[94]. This formula is correct for
oligonucleotides with a length between 14 and 70 nucleotides.
This formula calculates T, the optimal annealing temperature
psy177 2-5psy176 C. 
is the effective length of the primer:
[95].
This equation should result in correct values for oligonucleotides with 20 to
35 nucleotides.