Sequence
Primer and Tm
Melting temperature from the nearest-neighbour model with salt and magnesium correction, plus hairpin, self-dimer and primer-dimer checks and a suggested annealing temperature.
Reaction conditions
Tm uses the nearest-neighbour model (SantaLucia 1998) with salt correction; free magnesium is magnesium minus dNTP. The suggested annealing temperature is five degrees below the lower Tm and is a starting point, not an answer — run a gradient for each new primer pair.
Formula
Tm = ΔH° ÷ (ΔS° + R·ln(C_T/4)) − 273.15
- ΔH°
- summed enthalpy of adjacent base pairs, kcal/mol
- ΔS°
- summed entropy, after salt correction
- R
- gas constant, 1.987 cal/mol·K
- C_T
- total oligo concentration; divided by four for a non-self-complementary primer
- [Na⁺]eq
- equivalent sodium = sodium + 120 × √(magnesium − dNTP)
Worked example
The M13 forward primer GTAAAACGACGGCCAGT under standard conditions
- length = 17 nt, GC = 52.9%
- primer concentration = 250 nM
- [Na⁺] = 50 mM, no magnesium
- For each of the sixteen adjacent base pairs, enthalpy and entropy are read from the SantaLucia table and summed.
- An initiation term is added for both ends; a G or C end contributes differently from an A or T end.
- Entropy is corrected by 0.368 × (length − 1) × ln([Na⁺]eq).
- The values go into the equation above and kelvin is converted to celsius.
Answer about 51.7 °C — where the simple 4+2 rule gives 47.1 °C
Common mistakes
- Using 2(A+T) + 4(G+C) for primers longer than thirteen nucleotides. That rule was built for short oligos under particular conditions and is several degrees off for PCR primers.
- Leaving magnesium out. Magnesium screens the negative phosphate backbone and raises Tm by several degrees; ignoring it means underestimating your annealing temperature.
- A large Tm gap between the two primers of a pair. Beyond about three degrees, one primer anneals poorly at a single annealing temperature while the other binds non-specifically.
- More than three G or C in the last five bases at the 3′ end. The 3′ end becomes too stable and locks in mispriming.
- Treating the annealing temperature as equal to Tm. It is usually set about five degrees lower, and every new primer pair deserves a gradient.
Frequently asked
Why are two different Tm values shown?
The nearest-neighbour column uses the thermodynamics of adjacent base pairs together with your actual reaction conditions and is the accurate one. The simple-formula column is kept only for comparison with older protocols and papers that quote it.
How much does a hairpin matter?
A stem shorter than four bases usually melts at the annealing temperature and can be ignored. Five bases or more, especially near the 3′ end, measurably lowers amplification efficiency.
What about a primer with an added tail?
In the first cycles only the template-matching part anneals, so calculate Tm for that part. A restriction site or tag becomes part of the product from the third cycle onward and raises the effective Tm.