PCR Product Smearing on Gel (End-point PCR Primers)

Question

Accepted Answer

Gel electrophoresis shows smeared or streaked bands instead of distinct sharp bands Common causes: 1) Excessive enzyme, primer, Mg2+, or dNTP concentration 2) Annealing temperature too low for primers 3) Too many PCR cycles performed 4) Annealing and extension times too long 5) Degraded or old primers 6) Excessive template (>10^6 molecules initially) Solutions: 1) Reduce concentrations of enzyme, primers, Mg2+, and dNTPs 2) Increase annealing temperature 3) Decrease number of PCR cycles 4) Shorten annealing and extension times 5) Use fresh primers 6) Start with 10^4–10^6 template molecules 7) Consider using PCR Optimizer Kit if primers previously worked with Taq