Endpoint PCR Troubleshooting

PROBLEM

CAUSES

SOLUTIONS

No PCR Product or very low yield

Detection failure

• Recheck electrophoresis conditions and performance of the dye used for the gel staining. If there are suspicions that this can be detection problem, run the gel once more, load more PCR product on the gel, if possible. Make sure that the gel is stained correctly and the DNA ladder is well visible.
• For approximate quantification of the PCR product a standard agarose gel electrophoresis and ethidium bromide staining is most commonly used. By using appropriate DNA Ladders and DNA Loading Dyes one can both determine the size of the PCR product and approximately quantify the DNA by comparing the intensity of the band with a closest size DNA band of the ladder of the well known quantity.
• If the PCR is performed with standard reagents that do not contain dyes, add DNA loading dye to reactions prior loading onto the gel. To save time and effort, special enzymes, buffer and master mix formulations that contain electrophoresis loading dyes can be used to allow loading the PCR products directly onto the gel.
• When performing electrophoresis, it is generally advisable to load different volumes (2–10µl) of the reaction onto the gel. Loading insufficient reaction volumes might incorrectly be interpreted as no yield in case of low yields. Loading excessive reaction volumes might cause sizing and quantification problems in case of high yields.

Impurities inhibiting PCR

• Inhibitors coming from template, primers and low-quality reagents can reduce PCR yield. Purify template DNA and use highest quality reagents for PCR, especially dNTPs and primers, from reliable a supplier.
• Trace amounts of contaminants remaining sometimes after DNA purification, such as phenol, EDTA or proteinase K, can inhibit PCR. Remove contaminants with ethanol precipitation and wash the DNA pellet with 70% ethanol.
• Include a positive control reaction for each experiment to help to identify the source of inhibiting impurities.To determine whether a questionable template preparation is inhibiting PCR, add template and primers that are known to work for a new reaction. Compare result with the control reaction that does not contain the questionable template. Similar controls can be used to test primers and other reagents for PCR inhibition.

Inapropriate or low quality primers

• If possible, use dedicated software for primer design. Software will help you to avoid primer self-complementarities, multiple template–primer complementarities, and direct repeats in the primer to prevent hairpin and primer–dimer formation. Complementarity at the 3' ends causes the formation of primer–dimers and is one of the most common PCR problems.
• Design primers of 20–30 nucleotides in length. Ideally, use primers with about 50% GC content. If possible, choose primers with one or two Gs or Cs at the 3’ end.
• The purity and chemical integrity of the primers is crucial for good PCR results. Primers should always be present in excess in the reaction mixture and at equal concentrations, typically within the range of 0.1 μM to 1 μM. Optimal primer concentration is determined empirically. Excessive primer concentration can reduce PCR specificity due to increased miss-priming.

Low quality template, low abundance or complex template

• Purify genomic DNA carefully and avoid sharing. Use purification kits designed for the type of template DNA. Optimal amounts of template DNA for a 50 µl reaction volume:

          Plasmid or phage: 0.01–1 ng
          Genomic: 0.1–1 µg

• Excessive amounts of template increase nonspecific PCR. Insufficient amounts of template reduce fidelity. Use hot-start enzymes for higher sensitivity when template abundance is low. For complex or GC-rich templates, use additives and more enzyme accordingly or use products developed for GC-rich PCR.

Low quality dNTPS, wrong concentration of dNTPs

• For the best PCR results, use only highest quality, 99% pure dNTPs. dNTP purity is especially important for long-range and high-fidelity PCR.
  
• Unbalanced dNTP concentrations can result in reduced fidelity.The recommended final concentration for each dNTP is 0.2 – 0.25 mM. Higher concentration (up to 0.4 mM) may increase yields.
  
• Due to the binding of Mg²⁺ to dNTPs, the Mg²⁺ concentration should be adjusted accordingly. Excessive dNTP and magnesium concentrations reduce PCR fidelity.Thaw and mix each dNTP solution before use. This is especially important for high-concentration solutions.

Suboptimal magnesium concentration

• Magnesium ions stabilize primer–template complexes. Some PCR buffers for Taq DNA Polymerase contain Mg²⁺ at optimal concentration. Other buffers do not contain magnesium (Taq DNA Polymerase, recombinant) but are provided with a separate vial of magnesium chloride, allowing different magnesium concentrations to be prepared.
• Mg²⁺ binds to dNTPs, primers and DNA template. It is highly recommended that Mg²⁺ concentration (in a range of 1–4 mM) is optimized for every PCR.
• Insufficient magnesium reduces the yield of PCR product. Excessive magnesium concentration causes non-specific PCR products and reduced PCR fidelity.
• The ratio of dNTPs and magnesium in the PCR should generally be 1:2. When dNTP concentration is increased, the magnesium concentration should be increased accordingly.
• If DNA samples contain EDTA or other metal chelators, the magnesium ion concentration should be increased accordingly, as one molecule of EDTA binds one molecule of Mg²⁺.
• Many applications work at the standard concentration of 1.5 mM MgCl₂. However advanced applications using genomic DNA require higher MgCl₂ concentrations (2–4 mM). Adjust PCR magnesium concentration with the supplied MgCl₂ solution according to the table below:

Suboptimal cycling

• Complete initial DNA denaturation is essential for good PCR yield. Typically 1–3 minutes denaturation at 95°C is sufficient. For GC-rich templates, up to 10 minutes might be needed. If a very long initial denaturation step is performed, it is advised to add the enzyme only after the denaturation is completed.
• During cycling, the denaturation  is typically performed 30 seconds to 2 minutes at 94–95°C per cycle. For GC-rich templates, this step can be prolonged to 3–4 minutes. Alternatively, DNA denaturation can be enhanced by using additives, such as glycerol, DMSO or formamide. When additives are used, the annealing temperature must to be adjusted, as the melting temperature of the primer–template complexes might decrease significantly.

• Calculate primer melting temperatures using appropriate software. Primer annealing temperature should be 5°C lower than the lowest primer melting temperature.The annealing duration is typically 30 seconds to 2 minutes.
• Low annealing temperature leads to high background. High annealing temperatures might reduce yield.
• Optimize annealing temperature stepwise in 1–2°C increments.
• In the presence of additives (glycerol, DMSO, formamide and betaine), the annealing temperature may need to be adjusted.

• During PCR extension step, the primer elongation reaction is typically performed for 1 minute at 72°C, the optimal temperature for Taq DNA Polymerase, for PCR products up to 2 kb.
• For long-range PCR, the extension time is prolonged by 1 min/kb and extension temperature is lower (68^oC) to minimize loss of enzyme activity.
• As Pfu is slower than Taq DNA Polymerase, an extension step of 2 minutes per kb at 72°C is used.
• The final extension step is usually longer to ensure completion of PCR products. This final step, typically 15–30 minutes at 72^oC, is especially important when the PCR product will be cloned into the T vector.

Insufficient PCR cycles

• The optimal number of cycles should be determined according to the quantity and complexity of the template and the amplicon length.Typically, 25–30 cycles are enough for a good yields.
• An insufficient number of cycles may result in poor yield.
• Too many cycles can cause high background.
• When the amount of template is very low, 10 copies for example, 40 cycles is recommended.

Incorrect products used for PCR

• Select an optimal product to meet your PCR needs. Follow the Product Selection Guides to choose the right product:

• Cloning related applications    Endpoint PCR Technique Selection Guide
• Standard PCR    Application overview and Product Selection Guide
  
• Hot Start PCR    Application overview and Product Selection Guide
• High Fidelity, Long and GC Rich PCR   Application overview and Product Selection Guide
• Reverse Transcription and RT PCR    Application overview and Product Selection Guide

Short or long unspecific products

Primer dimers, faulty primer design

• If possible, use dedicated software for primer design. Software will help you to avoid primer self-complementarities, multiple template–primer complementarities, and direct repeats in the primer to prevent hairpin and primer–dimer formation. Complementarity at the 3' ends causes the formation of primer–dimers and is one of the most common PCR problems.
• Design primers of 20–30 nucleotides in length. Ideally, use primers with about 50% GC content. If possible, choose primers with one or two Gs or Cs at the 3’ end.
• The purity and chemical integrity of the primers is crucial for good PCR results. Primers should always be present in excess in the reaction mixture and at equal concentrations, typically within the range of 0.1 μM to 1 μM. Optimal primer concentration is determined empirically. Excessive primer concentration can reduce PCR specificity due to increased miss-priming.
• Use Hot Start Enzymes to minimize primer dimer formation during room temperature reaction set up.

Excessive magnesium

• Excessive magnesium concentration causes non-specific PCR products and reduced PCR fidelity.
• Some PCR buffers for Taq DNA Polymerase contain Mg²⁺ at optimal concentration. Do not add more magnesium into such buffers.
  
• Other buffers do not contain magnesium (Taq DNA Polymerase) but are provided with a separate vial of magnesium chloride, allowing different magnesium concentrations to be prepared.
• It is highly recommended that Mg²⁺ concentration (in a range of 1–4 mM) is optimized for every PCR.
• Many applications work at the standard concentration of 1.5 mM MgCl₂. However advanced applications using genomic DNA require higher MgCl₂ concentrations (2–4 mM).
  
• Adjust PCR magnesium concentration with the supplied MgCl₂ solution as described  above in this table.
  

Excessive template

• Optimal amounts of template DNA in the 50 µl reaction volume are:

              in the 0.01-1 ng range for plasmid or phage DNA
              in the 0.1-1 µg range for genomic DNA

• Choose the right template amount for every reaction empirically.Too high amounts of template increase nonspecific PCR, too low amounts of template reduce the accuracy.

Room temperature reaction set up

• Assemble the PCR reaction on ice, keep all reagents on ice.
• For room temperature set up use only Hot Start Enzymes. Thermophilic polymerases are active at room temperature, which results in amplification of unspecific targets due to random primer annealing events. Hot Start Enzymes are completely inactive during room-temperature reaction setup and are activated only after heating. Thus, miss-primed amplification and primer–dimer formation does not occur, diminishing the PCR background.
  

Suboptimal annealing  conditions

• Primer annealing temperature should be 5°C lower than the lowest primer melting temperature (Tm).
  
• The time of annealing is 30 sec – 2 min. Too low annealing temperature leads to high background, too high one may lead to no product in PCR.

Diffused, smeared band on the gel

Detection failure

• Recheck electrophoresis conditions and performance of the dye used for the gel staining. There might be nuclease contamination in the electrophoresis or staining buffer. If there are suspicions that this can be detection problem, run the gel once more, load more PCR product on the gel, if possible. Make sure that the gel is stained correctly and the DNA ladder is well visible.
• When performing electrophoresis, it is generally advisable to load different volumes (2–10µl) of the reaction onto the gel. Loading insufficient reaction volumes might incorrectly be interpreted as no yield in case of low yields. Loading excessive reaction volumes might cause sizing and quantification problems in case of high yields.

Impure, degraded template

• Purify genomic DNA carefully and avoid sharing. Use purification kits designed for the type of template DNA. Optimal amounts of template DNA for a 50 µl reaction volume:

• Excessive amounts of template increase nonspecific PCR. Insufficient amounts of template reduce fidelity.

Low PCR specificity

• Use Hot Start Enzymes for PCR to increase specificity and follow the same recommendations like above for Short or long unspecific products.

Negative control is “positive”

PCR contamination

• Keep a clean work environment. When preparing PCR, care should be taken to eliminate the possibility of contamination with foreign DNA templates.
• Use separate clean areas for sample preparation, reaction mixture preparation and cycling.
• Before PCR setup, clean your working space with 70% ethanol or with a special DNA-removing spray.
• Always wear fresh gloves.
• Use sterile tubes and pipette tips with aerosol filters for PCR setup. Most contaminating templates come from dirty pipettes.
• Use DNA- and nuclease-free reagents, including water.
• Use the advantages of Uracil-DNA Glycosylase: Amplification of contaminating templates remaining in the work environment from previous experiments is one of the most common contamination problems. The use of Uracil-DNA Glycosylase (UDG or UNG) and dUTP (dNTP/dUTP Mix) helps prevent carry-over contamination.The typical workflow follows:

  Uracil-DNA Glycosylase degrades DNA containing uracil. To prevent carry-over contamination, use dUTP or dTTP/dUTP rather than dTTP alone for PCR. Taq DNA Polymerases incorporate dUTP efficiently. However, with proofreading polymerases, such as Pfu DNA Polymerase or Long and High Fidelity PCR Master Mix, the use of dUTP will result in lower PCR yield. dUTP does not change the electrophoretic mobility or ethidium-bromide-staining efficiency of DNA. After PCR with dUTP, the PCR products are substrates for UDG and any DNA containing uracil is degraded during the brief incubation with Uracil-DNA Glycosylase. This step eliminates contaminating DNA templates from previous experiments.

Unsuccessful cloning of PCR product

Incorrect preparation of the PCR product for cloning

• Always purify PCR products from the gel before cloning; otherwise, primer–dimers and unspecific products may be cloned.
• Avoid exposure to UV and over-staining with ethidium bromide when cutting out the PCR band from the gel. These factors increase mutation frequency and damage the integrity of DNA. Keep the gel on the glass plate or plastic during UV exposure or use dyes that do not require UV for detection.

Poor quality cloning reagents

• Use only high quality reagents for cloning, pure Ligase, reliable vector, high efficiency competent cells.

Incorrect cloning strategy

• Taq DNA Polymerases produce PCR products with A tails, which are suitable for TA cloning and can be cloned into T vectors without special treatment. For more efficient TA cloning, perform longer final PCR elongation step (up to 30 minutes) at 72°C.
• Pfu DNA Polymerase or Long and High Fidelity PCR Master Mix produce PCR products with blunt and overhanging ends. These products should be blunted prior to cloning into blunt-end vectors. High-fidelity PCR products are not suitable for TA cloning.

Too many sequence errors in PCR product

Incorrect choice of the enzyme

• Choose the correct PCR enzyme for the desired error rate.
  
• For higher fidelity, use Pfu DNA Polymerase or Long and High Fidelity PCR Master Mix. The expected error rate is typically indicated in enzyme description.

Excessive  magnesium in the reaction

•  Excessive magnesium concentration causes non-specific PCR products and reduced PCR fidelity.
• Some PCR buffers for Taq DNA Polymerase contain Mg²⁺ at optimal concentration. Do not add more magnesium into such buffers.
  
• Other buffers do not contain magnesium (Taq DNA Polymerase) but are provided with a separate vial of magnesium chloride, allowing different magnesium concentrations to be prepared.
• It is highly recommended that Mg²⁺ concentration (in a range of 1–4 mM) is optimized for every PCR.
• Many applications work at the standard concentration of 1.5 mM MgCl₂. However advanced applications using genomic DNA require higher MgCl₂ concentrations (2–4 mM).
  
• Adjust PCR magnesium concentration with the supplied MgCl₂ solution as described  above in this table.

Low quality, unbalanced dNTPs

• For the best PCR results, use only highest quality, 99% pure dNTPs. dNTP purity is especially important for long-range and high-fidelity PCR.
  
• Unbalanced dNTP concentrations can result in reduced fidelity.The recommended final concentration for each dNTP is 0.2 – 0.25 mM. Higher concentration (up to 0.4 mM) may increase yields.
  
• Due to the binding of Mg²⁺ to dNTPs, the Mg²⁺ concentration should be adjusted accordingly. Excessive dNTP and magnesium concentrations reduce PCR fidelity.Thaw and mix each dNTP solution before use. This is especially important for high-concentration solutions.

Too little template

• Optimal amounts of template DNA for a 50 µl reaction volume:
  

• Excessive amounts of template increase nonspecific PCR. Insufficient amounts of template reduce fidelity.

Low quality primers

• Use only reliable supplier for primers. Low quality primers may contain sequence errors.

DNA damage due to UV exposure

• Avoid exposure to UV and overstaining with ethidium bromide when cutting out the PCR band from the gel. This increases mutation frequency and damages the integrity of DNA. Keep the gel on the glass plate or plastic during UV exposure or use dyes that do not require UV for detection.

Sequencing errors

• Repeat sequencing reaction, in case of complex and GC rich DNA use dedicated sequencing reagents.

Results are not  reproducible

Low quality regents

For the best PCR results use high quality reagents:

• dNTP Sets and Mixes where 99% pure dNTPs are included.
• Use highly purified enzymes and nuclease free buffers for PCR.
• Recheck the quality and integrity of the template before every PCR if possible.
• Use only dedicated pipets and tubes of high quality plastic which is not binding the DNA or proteins.
  

Setup errors

• Mix well all reagents before every use.
• Make mastermixes whenever possible to reduce pipetting errors.

Component changes

• Monitor every change of the reagents or plastics which you use for PCR. Every change can influence PCR results.

Nuclease contamination

• Keep a clean work environment. When preparing PCR, care should be taken to eliminate the possibility of contamination with nucleases.
• Use separate clean areas for sample preparation, reaction mixture preparation and cycling.
• Before PCR setup, clean your working space with 70% ethanol or with a special spray.
• Always wear fresh gloves.
• Use sterile tubes and pipette tips with aerosol filters for PCR setup.
  
• Use DNA- and nuclease-free reagents, including water.