Frequently asked questions

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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 (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/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.


Categories:
Custom DNA & RNA qPCR & PCR

Tags:
oligo design oligoanalyzer oligonucleotide primers