General Guidelines for PCR Optimization

Use high quality, purified DNA templates whenever possible. Please refer to specific product information for amplification from unpurified DNA (e.g., colony PCR or direct PCR);
For low complexity templates (e.g., plasmid, virus, BAC DNA), use 0.001–1 ng of DNA per 50 μl reaction;
For higher complexity templates (e.g., genomic DNA), use 1–25 ng of DNA per 50 μl reaction;
Higher DNA concentrations tend to decrease amplicon specificity, particularly when a high number of cycles are run;

Primers should typically be 20–40 nucleotides in length;
Ideal GC primer content between 40–60%, however, we consider that the GC content parameter for primer evaluation is outdated and unnecessary;
Primer Tm and annealing temperatures (Ta) calculation should be determined with PrimerDigital’s Tools;
Polynucleotide stretches should be avoided, runs of three or more G residues in particular can cause problems due to intermolecular stacking;
Primer pairs should have Tm values that are within 3°C (Designing primers with the same dG will render more efficient primers pairs, matching Tm’s is a less accurate approach than matching dG’s.);
Avoid secondary structure (e.g., hairpins) within each primer and potential dimerization between the primers;
Final concentration of each primer should be 10–500 nM in the reaction (optimal 100–300 nM);
Higher primer concentrations may increase secondary priming and create spurious amplification products;
When amplifying products > 5 kb in size, primers should be ≥ 25 nucleotides in length and matched Tm values above 68°C;
When engineering restriction sites onto the end of primers, 6 nucleotides should be added 5´ to the site;

Optimal Mg²⁺ concentration is usually 1.5–2.0 mM for most PCR polymerases;
Most PCR buffers already contain sufficient levels of Mg²⁺ at 1X concentrations;
A variety of Mg-free reaction buffers to which supplemental Mg²⁺ can be added for applications that require complete control over Mg²⁺ concentration;
Further optimization of Mg²⁺ concentration can be done in 0.2–1 mM increments, if necessary. For some specific applications, the enzyme may require as much as 6 mM Mg²⁺ in the reaction;
Insufficient Mg²⁺ concentrations may cause reaction failure but excess Mg²⁺;

Ideal dNTP concentration is typically 200 μM of each, however, some enzymes may require as much as 400 μM each;
Excess dNTPs can chelate Mg²⁺ and inhibit the polymerase;
Lower dNTP concentration can increase fidelity, however, yield is often reduced;

Optimal enzyme concentration in the reaction is specific to each polymerase;
In general, excess enzyme can lead to amplification failure, particularly when amplifying longer fragments;

Optimal denaturation temperature ranges from 90°–98°C and is specific to the polymerase in the reaction;
Avoid longer or higher temperature incubations unless required due to high GC content of the template;
For most PCR polymerases, denaturation of 1–10 seconds is recommended during cycling;
XCR® a variant of PCR methods in which assay design and thermal amplification profile are approached. Rather than completely denature a DNA sample and expecting all of the nucleic acid in the sample to denature and fall apart, XCR® uses explicitly designed denature temperatures to minimize the possibility of amplification primers binding to non-target regions.
Aptamer-based hot start enzymes do not require additional denaturation steps to activate the enzymes;

Primer Tm and annealing temperatures (Ta) values should be determined using PrimerDigital’s Tools;
Non-specific product formation can often be avoided by optimizing the annealing temperature or by switching to a hot start enzyme;
Ta can be optimized by doing a temperature gradient PCR, starting at 5°C below the lowest Tm of the primer pair;
Ideally, primer Tm values should be near to the extension temperature. If Tm values are calculated to be greater than the extension temperature, a two-step PCR program (combining annealing and extension into one step) can be employed;
The most important values for estimating the Ta is Tm and the length of PCR fragment (L);
Primers with high Tm's (> 60°C) can be used in PCRs with a wide Ta range compared to primers with low Tm's (< 50°C);
The optimal annealing temperature for PCR is calculated directly as the value for the primer with the lowest Tm (T_m^min):

Extension temperature recommendations range from 65°–75°C and are specific to each PCR polymerase;
Extension rates are specific to each PCR polymerase. In general, extension rates range from 10–60 seconds per kb;
Longer than recommended extension times can result in higher error rates, spurious banding patterns and/or reduction of amplicon yields;

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