PCR Primer Tm Calculator
Calculate the melting temperature (Tm) of a PCR primer using the Wallace rule: Tm = 2(A+T) + 4(G+C).
How to Use the PCR Primer Tm Calculator
- Count each nucleotide in your primer sequence.
- Enter the number of Adenine (A) bases.
- Enter the number of Thymine (T) bases.
- Enter the number of Guanine (G) bases.
- Enter the number of Cytosine (C) bases.
- Click Calculate to see Tm, GC content, and primer length.
使用例
- •Designing PCR primers for gene cloning and expression studies.
- •Optimizing annealing temperatures for PCR protocols.
- •Comparing primer pairs for similar Tm values in multiplex PCR.
計算式
Wallace Rule: Tm = 2 × (A + T) + 4 × (G + C). Valid for primers up to ~20 bp. For longer primers use the nearest-neighbor method.
よくある質問
What is primer Tm?
The melting temperature (Tm) is the temperature at which 50% of the primer is annealed to its complementary sequence. It guides the choice of PCR annealing temperature, typically set 5°C below Tm.
When should I use the Wallace rule?
The Wallace rule is accurate for short oligonucleotides (< 20 bp) in standard salt conditions. For longer or complex primers, use nearest-neighbor thermodynamic calculations.
Disclaimer
For educational and research purposes only. Actual Tm values vary with salt concentration, primer concentration, and secondary structure. Experimental validation is recommended.