1. What is Oligo Annealing Temperature?

The oligo annealing temperature (T_ann) is the temperature at which primers optimally bind to the DNA template during PCR. The standard calculation is based on the Wallace rule which considers the number of G+C and A+T bases in the oligonucleotide.

2. How Does the Calculator Work?

The calculator uses the Wallace rule formula:

Where:

• \( G+C \) — Number of guanine and cytosine bases in the oligo
• \( A+T \) — Number of adenine and thymine bases in the oligo

Explanation: G-C base pairs have three hydrogen bonds while A-T pairs have two, so they contribute differently to the melting temperature.

3. Importance of Annealing Temperature

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

4. Using the Calculator

Tips: Count the number of G and C bases (G+C) and A and T bases (A+T) in your primer. Enter these values to get the estimated annealing temperature.

5. Frequently Asked Questions (FAQ)

Q1: Is this formula accurate for all oligos?
A: This is a simple estimation. For more accurate results, especially for longer oligos, consider using the nearest-neighbor method.

Q2: What's the typical range for annealing temperatures?
A: Most PCR primers work between 50-65°C, but optimal temperature depends on the specific primer sequence.

Q3: Should I use this exact temperature for PCR?
A: This is a starting point. Optimal temperature may need empirical testing, typically ±2-3°C from calculated value.

Q4: How does oligo length affect the calculation?
A: This simple formula works best for oligos 14-20 bases long. For longer oligos, more complex calculations are needed.

Q5: What about degenerate bases?
A: This calculator doesn't account for degenerate bases. For mixed-base oligos, use the lowest possible T_ann.