1. What is Oligo Melting Temperature?

The melting temperature (Tm) of an oligonucleotide is the temperature at which 50% of the DNA duplexes are denatured. The basic Wallace rule provides a simple method to estimate Tm based on nucleotide composition.

2. How Does the Calculator Work?

The calculator uses the Wallace rule equation:

Where:

• \( G \) — Number of guanine bases
• \( C \) — Number of cytosine bases
• \( A \) — Number of adenine bases
• \( T \) — Number of thymine bases

Explanation: The equation accounts for the fact that GC pairs (with three hydrogen bonds) contribute more to thermal stability than AT pairs (with two hydrogen bonds).

3. Importance of Tm Calculation

Details: Knowing the Tm is crucial for PCR primer design, hybridization experiments, and other molecular biology applications where DNA denaturation and renaturation are important.

4. Using the Calculator

Tips: Enter the count of each nucleotide in your oligonucleotide sequence. All values must be non-negative integers.

5. Frequently Asked Questions (FAQ)

Q1: How accurate is the Wallace rule?
A: It provides a rough estimate suitable for short oligonucleotides (15-20 bases). For more accurate results, consider more sophisticated methods that account for sequence context.

Q2: What are typical Tm values for PCR primers?
A: Most PCR primers are designed with Tm between 50-65°C, with forward and reverse primers typically within 5°C of each other.

Q3: Does salt concentration affect Tm?
A: Yes, higher salt concentrations increase Tm. The Wallace rule assumes standard conditions (50 mM NaCl).

Q4: What's the difference between Wallace rule and nearest-neighbor methods?
A: Nearest-neighbor methods consider the sequence context of each base pair and are more accurate but more complex to calculate.

Q5: Can I use this for RNA oligos?
A: This calculator is designed for DNA. RNA duplexes are generally more stable and would require different coefficients.