How do I design primers/probes for a Real-Time PCR assay?
There are numerous primer design tools commercially available for purchase or freely accessible via the web. These tools simplify assay design significantly. Some widely used primer design tools are Primer Express (Applied Biosystems), Beacon Designer (Premier Biosoft) and Real Time Design(BioSearch Technologies). Numerous websites contain databases of validated primer sets, including RTPrimerDB (http://medgen.ugent.be/rtprimerdb/). If you design primers and probes manually, follow these criteria from the Primer Express manual: • The amplicon length should be between 50-150 bp to maximize amplification efficiency. • The optimal primer length is 20 nucleotides and the Tm should be between 58° and 60° C. • The optimal probe length is between 13 and 30 nucleotides for standard TaqMan probes and the Tm should be between 68° and 70°C. • Primer and probe sequences should not overlap. • The GC content of primers and probes should be between 30% and 80%. • To prevent quenching, the 5′ end of t
