Look for PCR primers that conform to the following guidelines (use our free online OligoAnalyzer® tool for this purpose):
-> The difference between melting temperatures (Tm) of the primers should be less than 5°C.
-> The GC content should be between 35-80% or equivalent to the product being amplified. -> The Delta G value of any self-dimers, hairpins, and heterodimers should be weaker (more positive) than -9.0 kcal/mole. Positive numbers indicate that the actual secondary structure shown will not form at all.
-> Avoid 3' complementarity between the two primers to prevent primer dimers.
The IDT OligoAnalyzer program can be used to assess these different criteria for a proposed oligo.