Читать книгу PCR – diagnostics - Aizhan Zhussupova - Страница 4

Typical primers are 18-28 nucleotides with 50-60 % G+C content. Typical concentration in a PCR reaction is 0.1-0.5 mM; higher concentrations may give nonspecific products or primer dimers. Additionally, 3 or more C’s or G’s at 3’ ends of primers may promote mispriming at G+C rich sequences.

Furthermore, palindromic sequences within primers should be avoided; as should any internal inverted repeats that would cause primer to self anneal. Primers with a similar melting temperature that are completely complementary are recommended. The optimal melting temperature range for primers is 55 °C to 80 °C. An approximate melting value for your primer can be calculated before it is synthesized using the following equation: T_m (^oC) = 2(N_A+N_T) + 4(N_G+N_C).