PrimerDigital

General Guidelines for PCR Optimization

DNA Template

  • 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 1 pg–10 ng of DNA per 50 μl reaction;
  • For higher complexity templates (e.g., genomic DNA), use 1 ng–100 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

  • Primers should typically be 20–40 nucleotides in length;
  • Ideal primer content between 40–65% GC;
  • Primer Tm and annealing temperatures (Ta) calculation should be determined with PrimerDigital’s Tools;
  • Primer pairs should have Tm values that are within 5°C;
  • Avoid secondary structure (e.g., hairpins) within each primer and potential dimerization between the primers;
  • Final concentration of each primer should be 0.05–1 μM in the reaction (optimal 0.2–0.3 μM);
  • Higher primer concentrations may increase secondary priming and create spurious amplification products;
  • When amplifying products > 10 kb in size, primers should be ≥ 25 nucleotides in length with a GC content above 55% and matched Tm values above 65°C;
  • When engineering restriction sites onto the end of primers, 6 nucleotides should be added 5´ to the site;

Magnesium Concentration

  • 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 μM 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+;

Deoxynucleotides

  • 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;

Enzyme Concentration

  • 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;

Denaturation

  • Optimal denaturation temperature ranges from 94°–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 5–30 seconds is recommended during cycling;
  • Aptamer-based hot start enzymes do not require additional denaturation steps to activate the enzymes;

Annealing

  • 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 CG% of the primers 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 (Tmmin):
  • Ta calculation
    where L is length of PCR fragment.

Extension

  • 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;


Protocols

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