General guidelines for PCR optimization

Starting points for template amount, primer properties, buffer composition and cycling conditions — adjust them against your own polymerase's datasheet.

DNA template

  • Use high quality, purified DNA templates whenever possible. 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  • Ideal primer content is between 40–60% GC; however, we consider the GC content parameter to be outdated.
  • The last six bases of the 3′-end of the hybridizing part of the primer should be up to 50% GC content; the bases toward the 5′-end of the hybridizing part should be at least 60% GC content.
  • Primer Tm and annealing temperatures (Ta) should be determined with PrimerDigital's WebTools.
  • Primer pairs should have Tm values within 3°C. Designing primers with the same ΔG renders more efficient primer pairs; matching Tm is a less accurate approach than matching ΔG.
  • 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 larger than 5 kb, 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, add 6 nucleotides 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 Mg2+ at 1× concentration.
  • Mg-free reaction buffers are available, to which supplemental Mg2+ can be added for applications requiring complete control over Mg2+ concentration.
  • Further optimization can be done in 0.2–1 mM increments. For some applications the enzyme may require as much as 6 mM Mg2+.
  • Insufficient Mg2+ may cause reaction failure; excess Mg2+ reduces specificity.

Deoxynucleotides

  • Ideal dNTP concentration is typically 200 μM of each, though 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, but yield is often reduced.

Enzyme concentration

  • Optimal enzyme concentration 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 90–98°C and is specific to the polymerase in the reaction.
  • Avoid longer or higher temperature incubations unless required by high GC content of the template.
  • For most PCR polymerases, denaturation of 1–10 seconds is recommended during cycling.
  • XCR® is a variant of PCR in which assay design and the thermal amplification profile are approached differently: rather than completely denature a DNA sample and expect all of the nucleic acid to fall apart, XCR® uses explicitly designed denaturation 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 enzyme.

Annealing

  • Primer Tm and annealing temperature (Ta) values should be determined using PrimerDigital's WebTools.
  • 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 with a temperature gradient PCR, starting at 5°C below the lowest Tm of the primer pair.
  • Ideally, primer Tm values should be near 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 used.
  • The most important values for estimating Ta are the Tm and GC% of the primers and the length of the PCR fragment (L).
  • Primers with high Tm (> 60°C) can be used across a wider Ta range than primers with low Tm (< 50°C).
  • The optimal annealing temperature is calculated directly from the primer with the lowest Tm (Tmmin).
Formula for calculating the optimal annealing temperature from the lowest primer Tm and the PCR fragment length
where L is the length of the 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 they range from 10–60 seconds per kb.
  • Longer than recommended extension times can result in higher error rates, spurious banding patterns and/or reduced amplicon yields.

Protocols

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