How to Calculate Annealing Temperatures

Written by samuel sohlden
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How to Calculate Annealing Temperatures
A single strand of DNA, which represents an essential concept of PCR reactions (DNA image by chrisharvey from

Annealing temperature calculation is crucial for the success of a Polymerase Chain Reaction (PCR) procedure. This is because a precise temperature is required for the DNA primer to attach to the DNA sample. The primer allows for DNA replication by attaching complementary nucleotides to the single stranded DNA, which effectively doubles a given DNA sample, allowing for a small DNA sample to be transformed into a sample large enough for analysis purposes, such as in forensics. This entire process can fail or produce sub-par results if the correct annealing temperature is not used.

Skill level:

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Things you need

  • Primer (or primers)

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  1. 1

    Find or determine the nucleotide sequence of the primer or primers that will be used in the PCR reaction.

  2. 2

    Count the number of each type of nucleotide in the primer sequence. For example in a twenty nucleotide long single strand of DNA primer there could be five thymine, seven adenine, four cytosine, and four guanine.

  3. 3

    Plug the nucleotide numbers into this equation:

    Temp = 4 (G + C) + 2 (A + T) °C

  4. 4

    Calculate after inserting values. T:5, A:7, C:4, G:4

    Temp= 4 (4 + 4) + 2 (7 + 5) C

    Temp = 32 + 24 = 13.3 degrees C Celsius

  5. 5

    Subtract the calculated temperature by two to five degrees Celsius in order to determine the annealing temperature for the primer in the PCR reaction. This is necessary since the calculation in the previous step results in the melting temperature for a primer. The annealing temperature of a primer is typically two to five degrees Celsius lower than its melting temperature. According to the University of Capetown, "One should aim at using an annealing temperature (Ta) about 5 degrees Celsius below the lowest Tm of the pair of primers to be used."

  6. 6

    Subtract two to five degrees Celsius from the primer with the lowest melting temperature that will be used in PCR. Typically, two different primers are used in the PCR procedure, so it is essential to determine which primer has the lowest melting temperature in order to calculate the correct annealing temperature.

Tips and warnings

  • If the nucleotide sequence was not included with the primer, simply contact the company for the required information.
  • The formula listed above calculates primer melting temperature. Annealing temperature is typically two to five degrees Celsius lower than the melting temperature, making it necessary to subtract two to five degrees Celsius for the final answer.
  • PCR is a procedure typically performed in high school or college level laboratories. It can involve the usage of potentially cancerous chemicals, making it necessary for students to follow proper laboratory safety procedures.

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