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The page below is a sample from the LabCE course PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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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