Amplification Failure from Suboptimal Cycling Parameters (PCR (Invitrogen Guide))

Question

Accepted Answer

No product or very weak bands despite proper template and primer quality. Reaction components appear functional in other assays. Common causes: 1) Denaturation time/temperature insufficient to separate double-stranded DNA or too high reducing enzyme activity 2) Annealing temperature not optimized (should be 3–5°C below lowest primer Tm) 3) Extension time inadequate for amplicon length 4) Too few PCR cycles (generally need 25–35 cycles, up to 40 for <10 DNA copies) Solutions: 1) Optimize denaturation time/temperature balancing strand separation and enzyme stability 2) Optimize annealing temperature stepwise in 1–2°C increments using gradient cycler; adjust when using additives 3) Select extension time suitable for amplicon length; reduce to 68°C for long targets (>10 kb) 4) Adjust cycles to 25–35 (extend to 40 for low-copy templates); use high-processivity polymerases for short extension times