top of page

PCR primer design guidelines


  • Optimal primer length: 18-22 bases

  • Primer annealing temperature (Tm): 55C – 70C

  • GC content: 40-60%

  • GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.

  • Also, avoid primer secondary structures of high melting temperature, self-dimers, heterodimers between primer pairs, repeated sequences and large differences in annealing Tm (>5C) between primer pairs

Useful resources:

Primer design guidelines:

Tm Calculator for specific DNA polymerase brand products:!/  

bottom of page