PCR primer design guidelines

LAB PROTOCOL

• 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

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Useful resources:

Primer design guidelines: http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

Tm Calculator for specific DNA polymerase brand products: http://tmcalculator.neb.com/#!/