Table 1. A Summary of Primer Design Considerations.
| 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 that do not assume the secondary structure |
