# Annealing temperature FAQs

## Searching for the right temp?

Using the best annealing temperature is important for optimal hybridization and amplification and to avoid annealing to sequences other than the intended target. Review these FAQs for tips and tricks for finding the optimal annealing temperature for your oligos.

Whether you are annealing two complimentary oligos or carrying out a PCR reaction, calculating optimal annealing temperature is important to the success of your experiment. Below are some commonly asked questions about how to determine the best annealing temperature as well as how to anneal oligos, along with some tips to ensure you get the best results from your reaction.

## How do you determine annealing temperature?

When annealing complementary oligonucleotides from IDT to form a duplex, the standard temperature to use is 94°C for 2 minutes. If you are using primer pairs for PCR amplification, it is important to use the melting temperature (Tm) of the primers to determine the annealing temperature (Ta). The standard rule for determining annealing temperature is to set the Ta no more than 2–5°C below the lower Tm of the primers being used. To optimize the Ta, a formula can be used as outlined in the following FAQ.

## How do you calculate annealing temperature of primers?

To calculate the optimal Ta, the following equation can be used:

Ta Opt = 0.3 x (Tm of primer) + 0.7 x (Tm of product) – 14.9

Where the Tm of the primer is the melting temperature of the less-stable primer-template pair, and Tm of the product is the melting temperature of the PCR product [1].

## How do you set annealing temperature in PCR?

The annealing temperature (Ta) for PCR should be selected based on the Tm of the primers being used.  To avoid the chances of the primers annealing to sequences other than the intended target, select a Tthat is no more than 2﻿–5°C below the Tm of the primers being used (see above for the formula to calculate the Ta). This should be based on the lower primer Tm in the primer pair being used to find an optimal Ta because using a Ta that is too low can result in nonspecific amplification and a lower yield. Using a Ta that is higher than the Tm of the primers reduces the fraction of primer annealed to the target. To get the highest yield with the correct amplicon, it is important to use the correct annealing temperature.

IDT’s OligoAnalyzer™ Tool allows you to look up the Tm of any sequence and the PrimerQuest™ Tool allows you to check and compare the Tm of PCR primer pairs. Read this helpful article to better understand the importance of melting temperature in molecular biology applications and how to use it for oligo hybridization. IDT offers a variety of PCR products that can be used for your research needs.

## How do you calculate annealing temperature for PCR?

The annealing temperature for PCR can be calculated using the Ta Opt equation discussed earlier unless the primer pair has been previously optimized or the primer pairs have similar melting temperatures, in which case a Ta no more than 2⁠﻿–⁠5°C below the primer Tm can be used.

## How do you calculate annealing temperature from melting temperature (Tm)?

It is important to avoid setting the annealing temperature no more than 2⁠–5°C below the lower primer Tm as a general guideline. To optimize the Ta further, the optimization equation of Ta Opt = 0.3 x (Tm of primer) + 0.7 x (Tm of product) − 14.9, where Tm of primer is the melting temperature of the less stable primer-template pair and Tm of product is the melting temperature of the PCR product, can be used [1].

## How do you anneal oligos?

Annealing oligos involves taking single-stranded complimentary oligos and making double-stranded DNA. The protocol for annealing oligonucleotides from IDT includes resuspending the oligos in the appropriate duplex buffer at a high concentration, mixing the two oligos together in equimolar amounts, and annealing the two strands together. Heat the mixed oligos to 94°C for 2 minutes and cool gradually to room temperature. They can be diluted if needed but either way annealed oligos should be stored at −20°C.

## How do you check if oligos are annealed?

In order to see if the oligos have successfully annealed, they can be run on a non-denaturing gel. When run together with the appropriate molecular weight markers and the single-stranded non-duplexed oligos as controls, it should be apparent if the oligos successfully annealed. The double‑stranded band migrates more slowly compared to the single‑stranded band.

#### References

1. Rychlik W, Spencer WJ, Rhoads RE. Optimization of the annealing temperature for DNA amplification in vitro [published correction appears in Nucleic Acids Res 1991 Feb 11;19(3):698]. Nucleic Acids Res. 1990;18(21):6409-6412.
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Published Feb 6, 2024