How can I check my PCR primers using the OligoAnalyzer™ program to ensure there are no significant primer design issues?

Look for PCR primers that conform to the following guidelines (use our free online OligoAnalyzer Tool for this purpose):

• The difference between melting temperatures (T_m) 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/mol. 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.