Primer Design Summary Table - LabCE.com, Laboratory Continuing Education

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Primer Design Summary Table

Considerations	Comments
Primer melting temperature (Tm)	Tm = 4(G + C) + 2(A + T)°C
Primer length	18-22 bases
Primer annealing temperature (Ta)	Ta = 0.3 x Tm(primer) + 0.7 Tm (product) – 14.9
Percentage of GC content	40-60%
Tm difference between forward and reverse primers	Less than or equal to 5°C
Max 3’ stability	More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.
Verify primer not self complementary	< 4 contiguous bases
Not complementary to other primer(s)	< 4 contiguous bases
No long runs with the same bases	< 4 contiguous bases
No secondary structures	Identify primer pairs which do not assume secondary structure