Calculadora de Tm de Primer para PCR
Calcule a temperatura de anelamento ideal dos primers para otimizar suas reações de PCR.
Tm (Basic Formula)
56.7 °C
Tm (Basic Formula) vs Primer Length
Formula
## PCR Primer Melting Temperature Accurate Tm prediction is essential for PCR primer design. The salt-corrected formula accounts for ionic strength effects on DNA stability. ### Salt-Corrected Formula **Tm = 81.5 + 16.6 × log10([Na+]) + 41 × (GC fraction) - 675/N** where [Na+] is in moles/L and N is the primer length. The annealing temperature for PCR is typically set 3-5°C below the primer Tm.
Exemplo Resolvido
A 22-mer primer with 12 GC bases at 50 mM Na+.
- 01GC fraction = 12/22 = 0.5455
- 02Tm = 81.5 + 16.6 × log10(0.050) + 41 × 0.5455 - 675/22
- 03Tm = 81.5 + 16.6 × (-1.301) + 22.36 - 30.68
- 04Tm = 81.5 - 21.60 + 22.36 - 30.68 = 51.58°C
- 05Annealing temp ≈ 51.58 - 5 = 46.58°C
Perguntas Frequentes
What primer length and GC content are ideal?
Ideal primers are 18-25 bp long with 40-60% GC content, giving Tm between 55-65°C. Both primers in a pair should have Tm within 2°C of each other.
Should forward and reverse primers have the same Tm?
As close as possible, ideally within 2°C. Large Tm differences cause unequal binding efficiency, reducing PCR specificity and yield.
What about DMSO and formamide?
Additives that destabilize DNA reduce Tm. DMSO at 10% reduces Tm by about 5-6°C. Formamide reduces Tm by about 0.6-0.7°C per percent. Betaine helps equalize AT and GC melting.
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