What should be your starting temperature?

A common starting point is about 5 degrees below the melting temperature of the most labile primer, and usually in the range of 50 to 65 degrees Celsius. Use this as a center for your gradient and optimize from there.

Which melting temperature is more important?

Use the lower of the two temperatures. The melting temperature of the unstable primer is a factor in determining annealing temperature as it requires a low enough reaction temperature to bind. When designing primer pairs, make sure that the two melting temperatures are as close together as possible.

Why does GC content affect temperature?

Guanine and cytosine form three hydrogen bonds, while adenine and thymine are connected by two hydrogen bonds. Therefore primers with a high GC content bind less easily and require higher temperatures for binding.

What happens if the temperature for attachment is not suitable?

If the temperature is too low, primers will anneal to partially matching sequences, resulting in additional bands. If the temperature is too high, primers cannot anneal and little or no product is produced. A gradient PCR can quickly determine the optimal value.

Can I enter temperature in Fahrenheit or Kelvin?

Yes it is possible. You can change the unit on any temperature field. The calculator internally converts the temperature to celsius and applies the formula then displays the result in the selected unit.

Annealing Temperature Calculator

Design your PCR

How do you want to find the annealing temperature?

Product formula (Rychlik)

Two primers (Tm minus a margin)

From primer composition

Melting temperature of the less stable primer

Melting temperature of the product

Rychlik's product formula weights the primer and product melting temperatures. It is the most accurate of the three methods.

Annealing temperature (Tₐ)

PCR thermal cycle

Show the PCR thermal cycle

Set the denaturation and extension temperatures and chart the full cycle.

Denaturation temperature

Extension temperature

Annealing temperature (Tₐ)

°C

This sits in the usual 50 to 65°C window for annealing, a good place to start.

Gradient low: °C
Gradient high: °C

Not sure it is exactly right? Run a gradient PCR from 57.5°C to 63.5°C and keep the temperature that gives the cleanest band.

Charts and comparison

The annealing temperature is the temperature set in the PCR machine to allow primers to bind to template DNA. If it is set correctly only the target sequence will be amplified. A low temperature allows primers to bind at the wrong positions while a high temperature prevents them from binding at all.

This calculator provides three methods to determine this temperature: directly entering the melting temperature, calculating from values for two primers, and estimating based on primer length and GC content when the melting temperature is unknown.

What is annealing temperature in PCR?

PCR involves repeating several cycles, each of which consists of three temperature steps. First the reaction mixture is heated to about 95 degrees to separate double-stranded DNA into single strands. The temperature is then lowered to allow primers to anneal - that is, bind to their complementary sequences. Finally, the temperature is raised again, allowing polymerase to extend each primer and create a complete copy.

The annealing step is the most difficult part. The temperature must be low enough to allow primers to form base pairs with template DNA but high enough that they will only bind at fully complementary sites. This optimal range of temperatures varies from primer to primer, so it's more reliable to calculate than to intuitively guess.

Formula for product.

The most reliable estimate is based on an empirical formula published by Rychlik and colleagues in 1990. This formula combines the melting temperatures of the less stable primer and the amplified product.

T_\text{a} = 0.3 \times T_\text{m}^{\text{primer}} + 0.7 \times T_\text{m}^{\text{product}} - 14.9

If all temperatures are given in degrees Celsius, the constant 14.9 is used. This calculator does all calculations in degrees Celsius and then converts to the selected unit. So you can enter values in degrees Celsius, Fahrenheit or Kelvin and read them out without changing the formula.

For example, let's say we have a genetic target in cats. The less stable primer melts at 65.5 degrees while the product melts at 88.6 degrees.

T_\text{a} = 0.3 \times 65.5 + 0.7 \times 88.6 - 14.9 = 66.77\ ^\circ\text{C}

The calculation result is slightly above the usual temperature range for startup, which however does not represent a problem for an initial design and can be a good starting point for gradient experiments.

Calculation based on primers:

Often the melting temperatures of both primers are known but the product's melting temperature is unknown. As the less stable primer, i.e., the one with the lower melting temperature, often represents the limiting factor, a starting temperature some degrees below that temperature is set by a common rule of thumb.

T_\text{a} = \min\left(T_\text{m}^{\text{fwd}},\, T_\text{m}^{\text{rev}}\right) - \text{margin}

A 3 to 5 degree difference is standard. It is desirable for the melting temperatures of the two primers to be similar, ideally within a few degrees, so that one primer does not react too slowly during annealing.

Estimating Melting Temperature From Sequence.

If only the primer sequence is known, then the melting temperature can be estimated based on its length and GC content. This calculator offers three formulas, ordered from least to most realistic.

Formula	Equation	Best for
Basic (Wallace)	Tm = 4(G+C) + 2(A+T)	Short primers under 14 bases
GC content (Marmur)	Tm = 64.9 + 41(GC - 16.4)/N	Primers of 14 to 50 bases
Salt adjusted	Tm = 100.5 + 0.41(%GC) - 820/N + 16.6 log[Na+]	When salt matters

GC base pairs share three hydrogen bonds, while AT base pairs only share two. Therefore primers with a higher GC content have a higher melting temperature. Longer primers also have a higher melting temperature because more bonds need to be broken. Salts form a negatively charged shield around the DNA and therefore increase the melting temperature. For this reason, when using the salt correction formula, it is necessary to input the ion concentration.

Using the calculator tool:

Select a method above. For the product method, enter the melting temperatures of the most unstable primer and amplicon. For the two-primer method, enter the melting temperatures of both primers as well as their temperature difference. For the composition method, enter the length of the primer and number of G- and C-bases and select a formula to calculate the melting temperature.

The recommended annealing temperature is immediately displayed, along with a gradient range around that temperature. By opening the thermal cycle program panel you can set the denaturation and extension temperatures and graphically represent the complete temperature curve.

These estimates are only a starting point and not guaranteed. The melting temperature of primers can vary depending on the model used and reaction conditions so each primer pair should be tested in the lab. If unsure run a temperature gradient.

Frequently asked questions

What should be your starting temperature?: A common starting point is about 5 degrees below the melting temperature of the most labile primer, and usually in the range of 50 to 65 degrees Celsius. Use this as a center for your gradient and optimize from there.
Which melting temperature is more important?: Use the lower of the two temperatures. The melting temperature of the unstable primer is a factor in determining annealing temperature as it requires a low enough reaction temperature to bind. When designing primer pairs, make sure that the two melting temperatures are as close together as possible.
Why does GC content affect temperature?: Guanine and cytosine form three hydrogen bonds, while adenine and thymine are connected by two hydrogen bonds. Therefore primers with a high GC content bind less easily and require higher temperatures for binding.
What happens if the temperature for attachment is not suitable?: If the temperature is too low, primers will anneal to partially matching sequences, resulting in additional bands. If the temperature is too high, primers cannot anneal and little or no product is produced. A gradient PCR can quickly determine the optimal value.
Can I enter temperature in Fahrenheit or Kelvin?: Yes it is possible. You can change the unit on any temperature field. The calculator internally converts the temperature to celsius and applies the formula then displays the result in the selected unit.

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Disclaimer: This calculator is provided for general informational and educational purposes only. Our calculators are under active development, and results may be inaccurate, incomplete, or unsuitable for your situation. Always verify the figures independently and seek advice from a qualified professional before relying on them. We make no warranties and accept no liability for any loss or decision arising from use of this tool.

References

Rychlik W., Spencer W.J., Rhoads R.E. (1990): Optimization of the annealing temperature for DNA amplification in vitro
The primary paper behind the product annealing-temperature formula (Nucleic Acids Research 18(21):6409-6412).
Kibbe W.A. (2007): OligoCalc, an online oligonucleotide properties calculator
Source of the basic, GC-content, and salt-adjusted melting-temperature formulas (Nucleic Acids Research 35:W43-W46).
Wallace R.B. et al. (1979): Hybridization of synthetic oligodeoxyribonucleotides to Phi X 174 DNA
The 2(A+T) + 4(G+C) melting-temperature rule for short oligonucleotides (Nucleic Acids Research 6(11):3543-3557).