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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/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.