One of the single most important factors in successful PCR is primer design. It is important that a primer has the following characteristics:

 

A melting temperature (Tm) in the range of 52 °C to 65 °C

Absence of dimerization capability

Absence of significant hairpin formation (>3 bp)

Lack of secondary priming sites

Low specific binding at the 3' end (ie. lower GC content to avoid mispriming)

 

Primers designed according to these criteria will generally be from 18 to 30 bases in length and have %GC of 40 to 60. Try to avoid using primers with Tm's above 65-70oC, especially on high GC templates, as this can lead to secondary priming artifacts and noisy sequences. We strongly recommend the use of computer software to design primers with these characteristics. The following equation can be used to roughly estimate Tm:

 

Tm = 59.9 + 0.41*(%GC) - 600/length

 

If designing a primer based on existing sequencing data, choose a priming site that is more than 50 nucleotides away from the position where new sequence is needed. Avoid designing primers using regions of poorer quality sequence, such as areas beyond single peak resolution of a chromatogram (typically 600-700 bases). Avoid primers where alternative priming sites are present with more than 90% identity to the primary site or that match at more than seven consecutive nucleotides at the 3' end.

Finally, be aware that no set of guidelines will always accurately predict the success of a primer. Some primers may fail for no apparent reason, and primers that appear to be poor candidates may work well.