Insufficient Amplification from Polymerase Issues (PCR (Invitrogen Guide))

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Weak or absent PCR product. Primers are degraded or show primer-dimer formation at the bottom of gel. Common causes: 1) Proofreading polymerase 3′→5′ exonuclease activity degrading primers at room temperature 2) Insufficient DNA polymerase quantity for reaction requirements 3) Low Mg2+ concentration inadequate for polymerase activity 4) Excess PCR additives (DMSO, formamide) requiring more enzyme Solutions: 1) Use hot-start DNA polymerases to prevent primer degradation; alternatively set up PCR on ice or add polymerase last 2) Increase DNA polymerase amount per manufacturer recommendations, especially with high additive concentrations 3) Optimize Mg2+ concentration for maximum yield; increase if EDTA or high dNTPs present 4) Check polymerase preference for MgSO4 vs MgCl2 (e.g., Pfu works better with MgSO4) 5) Use lowest possible concentration of additives; adjust annealing temperature accordingly