1. What is Annealing Temperature?

The annealing temperature (Ta) is the temperature at which PCR primers bind to the template DNA. IDT (Integrated DNA Technologies) recommends calculating Ta as 5°C below the melting temperature (Tm) of the primer.

2. How Does the Calculator Work?

The calculator uses the IDT recommended formula:

Where:

• \( T_a \) — Annealing temperature (°C)
• \( T_m \) — Melting temperature of the primer (°C)

Explanation: The formula provides a general guideline for setting PCR annealing temperatures when using IDT primers.

3. Importance of Correct Annealing Temperature

Details: Proper annealing temperature is critical for PCR success. Too high may prevent primer binding, while too low may cause non-specific binding and amplification.

4. Using the Calculator

Tips: Enter the primer's melting temperature (Tm) in °C. The calculator will determine the recommended annealing temperature (Ta).

5. Frequently Asked Questions (FAQ)

Q1: How do I determine the Tm of my primer?
A: Tm can be calculated using IDT's OligoAnalyzer tool or other bioinformatics tools based on primer sequence.

Q2: Is this formula suitable for all PCR applications?
A: This is a general guideline. Some applications (like qPCR) may require optimization or different formulas.

Q3: What if my PCR isn't working with this Ta?
A: Consider optimizing in 2-5°C increments above or below the calculated Ta.

Q4: Does this work for both forward and reverse primers?
A: Yes, but when primers have different Tms, use the lower Tm for calculation.

Q5: What about PCR additives like DMSO?
A: Additives may affect optimal Ta. For each 10% DMSO, decrease Ta by 5-6°C.