What are the general considerations for PCR optimization?

There are several different types of general considerations for PCR optimization. 

• Use purified DNA templates when possible. For low complexity templates like plasmid or viral DNA, one should use 0.001–1 ng per reaction. For higher complexity templates like genomic DNA, one should use 1–25 ng per reaction. 
• Optimal Mg2+ concentration is usually 1.5–2.0 mM. Adjustments can be made in 0.2–1 mM increments if needed, but excess Mg2+ can inhibit the polymerase. Additionally, optimal enzyme concentration varies by polymerase. Excess enzymes can lead to amplification failure, especially with longer fragments. 
• Aim for primers 20–40 nucleotides long with an ideal GC content between 40–60% and to avoid polynucleotide stretches and secondary structures. The final concentration of each primer should be 10–500 nM in the reaction. Increased concentrations may lead to secondary priming. 
• Optimal denaturation temperature ranges from 90°–98°C, specific to the polymerase. One should keep denaturation times short (1–10 seconds) to avoid unnecessary damage. 
• Ideal dNTP concentration is typically 200 μM of each, though some enzymes may require up to 400 μM each. Excess dNTPs can inhibit the polymerase. 
• Extension temperature typically ranges from 65°–75°C, specific to the polymerase. Extension rates vary (10–60 seconds per kb), with longer times increasing error rates and spurious banding. One should also determine primer Tm and annealing temperatures using appropriate tools. Non-specific products can be minimized by optimizing annealing temperature or by using a hot start enzyme. 
• Optimal enzyme concentration varies by polymerase. Excess enzymes can lead to amplification failure, especially with longer fragments.