- 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.
|Complex targets (e.g., GC-rich or secondary structures)|
- 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.
|Other reaction components|
|Inappropriate DNA polymerase|
- 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.
|Insufficient quantity of DNA polymerase|
- 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.
|Insufficient Mg2+ concentration|
- 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.
|Excess PCR additives or co-solvents|
- 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).
|dUTP or modified nucleotides in reaction mix|
- 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.
|Thermal cycling conditions|
- 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 Tm.
- 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.
|Suboptimal number of PCR cycles|
- 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.