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• Minimize shearing and nicking of DNA during isolation. Evaluate template DNA integrity by gel electrophoresis, if necessary.
• Store DNA in molecular-grade water or TE buffer (pH 8.0) to prevent degradation by nucleases.

Low purity

• Follow manufacturer recommendations stringently when using purification kits to isolate template DNA. Consult the user manual and troubleshooting guides to mitigate poor DNA quality.
• Ensure that no residual PCR inhibitors such as phenol, EDTA, and proteinase K are present if following chemical or enzymatic DNA purification protocols.
• Re-purify, or precipitate and wash DNA with 70% ethanol, to remove residual salts or ions (e.g., K⁺, Na⁺, etc.) that may inhibit DNA polymerases.
• Choose DNA polymerases with high processivity, which display high tolerance to common PCR inhibitors carried over from soil, blood, plant tissues, etc.

• Examine the quantity of input DNA and increase the amount if necessary.
• Choose DNA polymerases with high sensitivity for amplification.
• If appropriate, increase the number of PCR cycles.

• Choose DNA polymerases with high processivity, which display high affinity for DNA templates and are more suitable to amplify difficult targets.
• Use a PCR additive or co-solvent to help denature GC-rich DNA and sequences with secondary structures.
• Increase denaturation time and/or temperature to efficiently separate double-stranded DNA templates.

• Check amplification length capability of the selected DNA polymerases. Use DNA polymerases specially designed for long PCR.
• Choose DNA polymerases with high processivity, which can amplify long targets in a shorter time.
• Reduce the annealing and extension temperatures to help primer binding and enzyme thermostability.
• Prolong the extension time according to amplicon lengths.

• Review primer design. Use online primer design tools when appropriate.
• Ensure that the primers are specific to the target of interest.
• Verify that the primers are complementary to the correct strands of the target DNA.

• Aliquot primers after resuspension and store properly.
• Reconstitute fresh primer aliquots, or obtain new primers if necessary.

• Optimize primer concentrations (usually in the range of 0.1–1 μM).
• For long PCR and PCR with degenerate primers, start with a minimum concentration of 0.5 μM.

• Use hot-start DNA polymerases to prevent degradation of primers by the 3’→5’ exonuclease activity of proofreading DNA polymerases. Hot-start DNA polymerases also increase yields of the desired PCR products by eliminating nonspecific amplification.
• Alternatively, set up PCR on ice, or add DNA polymerase last to the reaction mixture.

• Choose DNA polymerases with high sensitivity for amplification.
• Review recommendations on the amount of DNA polymerase to use in PCR, and optimize as necessary.
• Increase the amount of DNA polymerase if the reaction mixture contains a high concentration of an additive (e.g., DMSO, formamide) or inhibitors from the sample sources.

• Optimize Mg²⁺ concentration for maximum PCR yields. The presence of EDTA, other metal chelators, or atypically high concentrations of dNTPs may require a higher Mg²⁺ concentration.
• Check the DNA polymerase’s preference for magnesium salt solutions. For example, Pfu DNA polymerase works better with MgSO₄ than with MgCl₂.

• Review the recommended concentrations of PCR additives or co-solvents. Use the lowest possible concentration when appropriate.
• Adjust the annealing temperatures, as high concentrations of PCR additives or co-solvents weaken primer binding to the target.
• Increase the amount of DNA polymerase, or use DNA polymerases with high processivity.
• Consider using an additive or co-solvent specifically formulated for a given DNA polymerase (e.g., GC Enhancer supplied with Invitrogen Platinum DNA polymerases).

• Ensure that the selected DNA polymerases are able to incorporate the modified nucleotides.
• Optimize the ratio of the modified nucleotide to dNTP to increase PCR efficiency.

• Mix the reagent stocks and prepared reactions thoroughly to eliminate density gradients that may have formed during storage and setup.

• Optimize the DNA denaturation time and temperature. Short denaturing times and low temperatures may not separate double-stranded DNA templates well. On the other hand, long denaturation times and high temperatures may reduce enzyme activity.

• Optimize the annealing temperature stepwise in 1–2°C increments, using a gradient cycler when available. The optimal annealing temperature is usually 3–5°C below the lowest primer T_m.
• Adjust the annealing temperature when a PCR additive or co-solvent is used.
• Use the annealing temperature recommended for a specific DNA polymerase in its optimal buffer. Annealing temperature rules for primer sets can vary between different DNA polymerases.

• Select an extension time suitable for the amplicon length.
• Reduce the extension temperature (e.g., to 68°C) to keep the enzyme active during amplification of long targets (e.g., >10 kb).
• Use DNA polymerases with high processivity for robust amplification even with short extension times.

• Adjust the number of cycles (generally to 25–35 cycles) to produce an adequate yield of PCR products. Extend the number of cycles to 40 if DNA input is fewer than 10 copies.

• Review the optimal amounts of DNA input. Lower the quantity to reduce the generation of nonspecific PCR products.

• Degraded DNA may appear as smears or lead to high background in gel electrophoresis. Minimize shearing and nicking of DNA during isolation.
• Evaluate the integrity of the template DNA prior to PCR by gel electrophoresis, if necessary. Store DNA in molecular-grade water or TE buffer (pH 8.0) to prevent degradation by nucleases.

• Choose DNA polymerases with high processivity, which display high affinity for DNA templates and are more suitable to amplify difficult targets.
• Use a PCR additive or co-solvent to help denature GC-rich DNA and sequences with secondary structures. Increase denaturation time and/or temperature to efficiently separate double-stranded DNA templates.

• Check amplification length capability of the selected DNA polymerases. Use DNA polymerases specially designed for long PCR.
• Choose DNA polymerases with high processivity, which can amplify long targets in a shorter time.
• Reduce the annealing and extension temperatures to help primer binding and enzyme thermostability. Prolong the extension time according to amplicon lengths.

• Review primer design. Use online primer design tools when appropriate. Verify that the primers are specific to the target, with minimal homology to other regions in the template.
• Ensure that the primers do not contain complementary sequences or consecutive G or C nucleotides at the 3′ ends, to prevent primer-dimer formation.
• Avoid direct repeats in the primers to prevent misalignment in binding to the target. Consider longer primers to enhance specificity.
• Consider nested PCR to improve specificity.

• Optimize primer concentrations (usually in the range of 0.1–1 μM). High primer concentrations promote primer-dimer formation.

• Review recommendations on the amount of DNA polymerase to use in PCR, and decrease as necessary.

• Use hot-start DNA polymerases that have no activity at room temperature but are functional only after a high-temperature activation step, to enhance specificity. With non–hot-start DNA polymerases, set up PCR on ice to keep enzyme activity low.

• Review Mg²⁺ concentrations and lower as appropriate to prevent nonspecific PCR products. Optimize Mg²⁺ concentrations for each primer set and target DNA.

• Increase denaturation time and/or temperature to efficiently separate DNA when working with GC-rich templates and sequences with secondary structures.

• Use the annealing temperature recommended for a specific DNA polymerase in its optimal buffer. Annealing temperature rules for primer sets can vary between different DNA polymerases.

• Increase the annealing temperature to improve specificity. The optimal annealing temperature is usually no less than 3–5°C below the lowest primer T_m.
• Optimize the annealing temperature stepwise in 1–2°C increments, using a gradient cycler when available.
• Consider touchdown PCR to enhance specificity.

• Shorten the annealing time to minimize primer binding to nonspecific sequences.

• Reduce the extension temperature 3–4°C to help the DNA polymerase’s thermostability, especially for long PCR.

• Prolong the extension time when amplifying long DNA targets.
• Include a final extension step with sufficient time (5–15 minutes) to extend the whole target.

• Reduce the number of cycles, without drastically lowering the yield of the desired PCR products, to prevent accumulation of nonspecific amplicons.

• Use DNA polymerases with exceptionally high fidelity to generate PCR fragments for downstream applications such as cloning, sequencing, and site-directed mutagenesis.

• Review Mg²⁺ concentrations and reduce as necessary. Excessive concentrations favor misincorporation of nucleotides by DNA polymerases.

• Ensure equimolar concentrations of dATP, dCTP, dGTP, and dTTP in the reaction. Unbalanced nucleotide concentrations increase the PCR error rate.

• Reduce the number of cycles without drastically lowering the yield of the desired PCR products. High numbers of cycles increase the incorporation of mismatched nucleotides.
• Increase the amount of input DNA when appropriate to avoid running an excessive number of cycles.

• Use a long-wavelength UV (360 nm) light box to visualize fragments in gels, and limit the illumination time as much as possible.
• If using a short-wavelength (254–312 nm) light box, limit the UV illumination to a few seconds and keep the gel on a glass or plastic plate.
• Alternatively, use dyes with longer-wavelength (less damaging) excitation to visualize the DNA.

• Sequence both DNA strands to verify the reliability of sequencing results. Use duplicate samples when appropriate.

• Avoid direct repeats within primer sequences, as multiple repeats may appear from sequence misalignment at the ends of the PCR products.

• Order PCR primers with purification to remove non–full-length DNA oligos, which are truncated at their 5′ ends.

• Use molecular-grade, nuclease-free reagents in PCR setup. Set up reactions on ice to keep the activity of possible contaminating exonucleases low.

• Use a long-wavelength UV (360 nm) light box to visualize fragments in gels, and limit the illumination time as much as possible.
• If using a short-wavelength (254–312 nm) light box, limit the UV illumination to a few seconds and keep the gel on a glass or plastic plate.
• Alternatively, use dyes with longer-wavelength (less damaging) excitation to visualize the DNA.

• Sequence both DNA strands to verify the reliability of sequencing results. Use duplicate samples when appropriate.

• Avoid primers containing complementary and self-complementary sequences, which favor primer-dimer formation and self-oligomerization and their subsequent amplification.

• Avoid contamination of DNA in the work environment by following general recommendations for PCR setup.

• Use pipette tips with aerosol barriers. Dedicate a separate work area and decontaminate the area after each use.
• Follow PCR carryover control techniques such as dUTP incorporation with UDG treatment.