PCR Troubleshooting Guide - IL (2023)

Possible causesRecommendations
DNA templates
Poor integrity
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.
Insufficient quantity
Complex targets (e.g., GC-rich or secondary structures)
Long targets
  • 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.
Problematic design
  • 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.
Old primers
  • Aliquot primers after resuspension and store properly.
  • Reconstitute fresh primer aliquots, or obtain new primers if necessary.
Insufficient quantity
  • 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 0.5μM.
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.
Nonhomogeneous reagents
  • Mix the reagent stocks and prepared reactions thoroughly to eliminate density gradients that may have formed during storage and setup.
Thermal cycling conditions
Suboptimal denaturation
  • 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.
Suboptimal annealing
  • 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.
Suboptimal extension
  • 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.


How do you troubleshoot a PCR problem? ›

PCR Troubleshooting Guide
  1. Reduce number of cycles.
  2. Decrease extension time.
  3. Decrease Mg++ concentration in the reaction.

What are the common errors in PCR? ›

Many of the common problems with PCR and RT-PCR are identified during agarose gel electrophoresis of the reaction products. These include the absence of the expected amplification product, the presence of nonspecific products, excessive smearing, and the presence of a “primer dimer” band.

How do I make sure my PCR can run well? ›

Here are 13 ways of avoiding PCR errors when running PCR or any of its derivatives, including qPCR.
  1. 1 – Set Up a Sterile Environment. ...
  2. 2 – Check template purity and concentration. ...
  3. 3 – Take inventory of aliquoted PCR reagents. ...
  4. 4 – Make sure you choose the right annealing temperature.
Jul 9, 2020

What would cause PCR to fail? ›

The reason for PCR failure is usually rapidly identified. Often problems can be explained by the fact that essential components like Mg2+ ions or even primers were of poor quality because the expiration date has passed or were unintentionally not added to the reaction mix.

What happens if annealing temperature is too low in PCR? ›

Typically, the optimum annealing temperature is 3-5 degrees Celsius below the melting temperature. Too high of an annealing temperature prevents optimal binding of the primers to the templates while too low of an annealing temperature can lead to non-specific binding and, subsequently, non-specific PCR products.

Why am i getting multiple bands in PCR? ›

Too many PCR cycles (more than 30) also has the potential to cause multiple bands due to the increased chance of error with each cycle. DNA contamination is another possible factor.

What causes PCR accuracy error? ›

Thus, PCR accuracy reflects inaccuracies due to the granularity of the 27 MHz clock, and incorrect calculation of the inserted (or reinserted) value by a multiplexer (re-multiplexer).

Do PCR primers go bad? ›

yes, primers can go bad. However as mentioned, primers very rarely go bad... because there are so many primer molecules, all one experiences is a drop in product yields rather then absolute failure. The more likely cause of problems is template degredation.

What happens if you forgot to add primers in a PCR? ›

If you forgot to add the primers to your PCR reaction, what would happen and why? Your reaction would fail because Taq polymerase cannot add bases without a small piece of DNA already present. Your reaction would fail because there would be no enzyme that could add new nucleotide bases.

What causes smearing in PCR? ›

Excessively long extension times may result in smearing. The general recommendation for extension time for this enzyme is 10–20 sec/kb. If PCR yield is low, try increasing the number of cycles by 5.

What are the 5 key basic reagents used in PCR? ›

Generally, a complete polymerase chain reaction (PCR) requires five basic reagents, including DNA template, forward and reverse primers, DNA polymerase, deoxynucleotide triphosphates (dNTPs) and reaction buffer.

How do you optimize primers for PCR? ›

  1. Generally 20-30 nucleotides in length.
  2. Ideal GC content is 40-60%
  3. Space GC residues evenly within the primer.
  4. Calculated melting temperatures (Tm) should be from 42-65°C.
  5. Use the NEB Tm calculator to determine the optimal annealing temperature.
  6. Primer pairs should have Tms within 5°C of each other.

Is it possible to test negative for Covid PCR and still have it? ›

Conclusions: Our study showed that some patients with acute COVID-19 may test repeatedly negative by nasopharyngeal swab PCR. These cases should be interpreted as a low viral load in the upper respiratory tract rather than false negativity of PCR.

What does DMSO do in PCR? ›

DMSO is used to inhibit the secondary structure of DNA templates or DNA primers in polymerase chain reaction. It is added to the PCR mixture before the reaction, where it interferes with the self-complementarity of DNA and prevents interference reactions.

What causes primer dimers in PCR? ›

Causes of PCR/Primer Dimers in Sequencing Reactions

Contamination of the template, primer stock or other sequencing reagents with primer dimers. Too low an annealing temperature during the PCR. Two primer binding sites present in the template.

What are the sources of error for PCR and gel electrophoresis? ›

Answer and Explanation:

Some sources for gel electrophoresis include contamination of samples, using fluctuating voltages, and incorrect concentrations of matrix solid.

What are the sources of error in Qpcr? ›

Potential causes:
  • Pipetting error.
  • Insufficient mixing of solutions.
  • Low expression of target transcript resulting in stochastic amplification.
  • Poorly optimised reaction.
  • High Cq/low concentrations of template.
May 5, 2011

What is the error rate of PCR? ›

After 30 cycles of PCR amplifying a 3 kb template, only 3.96 % of the product DNA molecules contain 1 (nucleotide) error each. This means that 96.04 % of the product molecules are entirely error-free.

Are PCR false negatives common? ›

False negatives with PCR testing are actually far more common than one might expect. Daniel Rhoads, MD, vice chair of the College of American Pathologists microbiology committee who is also at the Cleveland Clinic, said PCR sensitivity for detecting COVID-19 is actually around 80%.

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