Melting temperature is an important factor in primer design. After the template sequence has been evaluated for free energy, SeqBuilder Pro can then determine melting temperatures for the template and subsequently located primers. PCR primer pairs which perform well have closely matched melting temperatures to promote amplification of both strands in equal amounts. The Tm of primers is calculated using entropy and enthalpy data from the nearest neighbor models:
…where salt is the concentration of Na+ in moles, R is the molar gas constant 1.987 cal/mol°C, and c is the initial concentration of the primer.
For long primers and probes (greater than 50 bases) and for products, a formula based upon GC content (Baldino et al. 1989) is used to calculate Tm.
Replace 0.35 (%formamide) with 0.50 (%formamide), as presented in Wahl et al..
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