Amplification Failure from Primer Problems (PCR (Invitrogen Guide))

Question

Accepted Answer

No visible PCR product despite good template quality. Control reactions with different primers may work normally. Common causes: 1) Primers not specific to target or complementary to wrong strand 2) Degraded or old primers with reduced binding efficiency 3) Primer concentration insufficient (below 0.1 μM range) 4) Primer design issues preventing proper target binding Solutions: 1) Verify primers are specific to target and complementary to correct strands using online design tools 2) Aliquot primers after resuspension; reconstitute fresh aliquots or obtain new primers 3) Optimize primer concentrations to 0.1–1 μM (minimum 0.5 μM for long PCR or degenerate primers) 4) Review and redesign primers ensuring proper Tm and target specificity