Low or No Amplification Due to Template Issues (PCR (Invitrogen Guide))

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PCR yields little to no product visible on gel electrophoresis. Expected band is absent or extremely faint despite using standard reaction conditions. Common causes: 1) DNA template degraded by shearing or nicking during isolation 2) PCR inhibitors (phenol, EDTA, proteinase K) carried over from purification 3) Residual salts or ions (K+, Na+) inhibiting DNA polymerase 4) Insufficient template quantity (fewer than minimum required copies) 5) GC-rich sequences or secondary structures preventing denaturation 6) Template longer than polymerase amplification capability Solutions: 1) Store DNA in molecular-grade water or TE buffer (pH 8.0) to prevent nuclease degradation 2) Re-purify or precipitate and wash DNA with 70% ethanol to remove salts and inhibitors 3) Use DNA polymerases with high processivity and tolerance to common inhibitors 4) Increase template quantity or number of PCR cycles (up to 40 cycles for <10 copies) 5) Use PCR additive or co-solvent to denature GC-rich DNA; increase denaturation time/temperature 6) Select DNA polymerases designed for long PCR with extended extension times