PCR Troubleshooting

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Overview

In conventional PCR, problems with reaction components and amplification protocols are diagnosed by running a gel. If you experience any of the symptoms pictured below when visualizing PCR products by agarose gel electrophoresis, click on the corresponding photo to learn about possible causes and treatments.

 

Related Topics: PCR Instruments, PCR Reagents, PCR Assay Design and Optimization, and PCR Analysis.

Problems and Solutions

Click on the image below that is most representative of your own gel to find out the probable cause and specific solutions to address your problem.

 

No Band or Faint Band

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Causes Related to Cycling Times and Temperatures
Too few cycles were used Using too few PCR cycles can lead to insufficient amplification. Use 20–35 cycles. Use fewer cycles when template concentration is high, and use more cycles when template concentration is low.
Extension time was too short If the extension time is too short, there will be insufficient time for complete replication of the target. Generally, use an extension time of 1 min/kb.
Annealing time was too short If the annealing time is too short, primers do not have enough time to bind to the template. Use an annealing time of at least 30 sec.
Annealing temperature was too high If the annealing temperature is too high, primers are unable to bind to the template. The rule of thumb is to use an annealing temperature that is 5°C lower than the Tm of the primer. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 µM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient. If the primer Tm minus 5°C is close to the extension temperature (72°C), consider running a two-step PCR protocol. The annealing temperature should not exceed the extension temperature.
Denaturation temperature was too low If the denaturation temperature is too low, the DNA will not completely denature and amplification efficiency will be low. Use a denaturation temperature of 95°C.
Denaturation time was too long If the denaturation time is too long, DNA might be degraded. For the initial denaturation, use 3 min at 95°C; for denaturation during cycling, use 30 sec at 95°C.
Denaturation time was too short If the denaturation time is too short, the DNA will not completely denature and amplification efficiency will be low. For the initial denaturation, use 3 min to activate the polymerase; to denature the template during cycling, use 30 sec.
Causes Related to PCR Components
dNTP concentration was too high If the dNTP concentration is too high, Mg2+ depletion occurs. Each dNTP should be present at 200 μM in the final reaction.
dNTP concentration was too low Each dNTP should be present at 200 μM in the final reaction.
PCR product has high GC content (>65%) GC-rich PCR products are difficult to amplify. To improve amplification, increase the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient. DMSO or another secondary structure destabilizer can be added (do not exceed 10%).
Template was damaged or degraded or contained inhibitors Template may be sheared or may contain PCR inhibitors. If inhibitors are suspected, dilute existing template; otherwise, use fresh template and increase cycles. Try a control reaction in which you use a pure plasmid with the addition of the template to determine if any inhibitory effects exist.
Primers contained impurities Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 μM of each primer.
Not enough template was in the reaction Insufficient amplification can result if the initial amount of template is too low. Increase the number of amplification cycles in increments of 5, or, if possible, increase the amount of template.
Impure dNTPs were used Contaminants in the dNTP mix can lead to incomplete or incorrect amplification or PCR inhibition. Use high-quality dNTPs.
Primer concentration was too high Using an excessive concentration of primers can increase the chance of primers binding nonspecifically to undesired sites on the template or to each other. Use well-designed primers at 0.2–1 μM in the final reaction. In addition, verify that the correct concentration was supplied by the manufacturer.
Primer concentration was too low If the primer concentration is too low, annealing may be inefficient. Use well-designed primers at 0.2–1 μM in the final reaction. In addition, verify that the correct concentration was supplied by the manufacturer.
Enzyme concentration was too low If the polymerase concentration is too low, not all PCR products will be fully replicated. The optimal enzyme concentration depends on the length and difficulty of the template.
Primers were designed or synthesized incorrectly by user or manufacturer Verify that primers have the correct sequence and are complementary to the template. Use a primer design program to avoid repetitive sequences, regions with high complementarity, etc. Perform a BLAST search to avoid primers that could amplify pseudogenes or that might prime unintended regions. Use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions) to calculate Tm. Use the lowest Tm of the primers.
Target was too long PCR component concentrations and/or cycling conditions may not be sufficient for longer target sequences. Reoptimize your existing assay protocol and/or increase the duration of PCR steps, especially the extension step.
Water was impure Water could have been contaminated during prior pipetting events. Use fresh nuclease-free water.
Not enough Mg2+ Insufficient or omitted magnesium will result in no or reduced PCR product. Use 1.5 mM in the final reaction.

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Causes Related to Omitted Components
dNTPs were omitted Each dNTP should be present at 200 μM in the final reaction.
Primer was omitted Use well-designed primers at 0.2–1 μM in the final reaction.
Template was omitted Add template.
Enzyme was omitted or inactive Use adequate units of enzyme. If you think the enzyme may be inactive, run a PCR with fresh polymerase from a different batch.
Mg2+ was omitted Insufficient or omitted magnesium will result in no or reduced PCR product. Use 1.5 mM in the final reaction.
 

Nonspecific Bands or Primer-Dimers

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Causes Related to Cycling Times and Temperatures
Too many cycles were used Excessive cycling increases the opportunity for nonspecific amplification and errors. Use 20–35 cycles. Use fewer cycles when template concentration is high, and use more cycles when template concentration is low.
Extension time was too long Excessive extension time can allow nonspecific amplification. Generally, use an extension time of 1 min/kb.
Annealing time was too long Excessive annealing time may increase spurious priming. Use an annealing time of 30 sec.
Annealing temperature was too low If the annealing temperature is too low, primers may bind nonspecifically to the template. The rule of thumb is to use an annealing temperature that is 5°C lower than the Tm of the primer. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient.
Thermal cycler ramping speed is too slow If the ramp speed of the cycler is too slow, spurious annealing may occur due to lower temperature and sufficient time for nonspecific binding. If ramping speed is not set at the maximum speed for the cycler, increase to maximum ramp rate.
Calculated primer Tm was inaccurate If the primer concentration is calculated incorrectly, the calculated annealing temperature will also be incorrect. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature.
Causes Related to PCR Components
Primers contain impurities Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 μM of each primer.
Too much primer was added Using a high concentration of primers may increase the chance of primers binding to nonspecific sites on the template or to each other. Use well-designed primers at 0.2–1 μM in the final reaction. In addition, verify that the correct concentration was supplied by the manufacturer.
Primers were designed or synthesized incorrectly by user or manufacturer Verify that primers have the correct sequence and are complementary to the template. Use a primer design program to avoid repetitive sequences, regions with high complementarity, etc. Perform a BLAST search to avoid primers that could amplify pseudogenes or that might prime unintended regions. Use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions) to calculate Tm. Use the lowest Tm of the primers.
Impure dNTPs were used Contaminants in the dNTP mix can lead to incomplete or incorrect amplification or PCR inhibition. Use high-quality dNTPs.
Too much Mg2+ was added Using high concentrations of magnesium increases the likelihood of nonspecific primer binding and unwanted product formation. Reduce the amount of magnesium in the final reaction.
Impure water was used Water could have been contaminated during prior pipetting events. Use fresh nuclease-free water.
 

Smeared Bands

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Causes Related to Cycling Times and Temperatures
Too many cycles were used Excessive cycling increases the opportunity for nonspecific amplification and errors. Use 20–35 cycles. Use fewer cycles when template concentration is high, and use more cycles when template concentration is low.
Extension time was too long Excessive extension time can allow nonspecific amplification. Generally, use an extension time of 1 min/kb.
Annealing time was too long Excessive annealing time may increase spurious priming. Use an annealing time of 30 sec.
Annealing temperature was too low If the annealing temperature is too low, primers may bind nonspecifically to the template. The rule of thumb is to use an annealing temperature that is 5°C lower than the Tm of the primer. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient.
Thermal cycler ramping speed is too slow If the ramp speed of the cycler is too slow, spurious annealing may occur due to lower temperature and sufficient time for nonspecific binding. If ramping speed is not set at the maximum speed for the cycler, increase to maximum ramp rate.
Calculated primer Tm was inaccurate If the primer concentration is calculated incorrectly, the calculated annealing temperature will also be incorrect. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 µM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature.
Causes Related to PCR Components
Too much template was added If the template concentration is too high, the polymerase can be inhibited due to carryover of inhibitors or inefficient denaturation. Reduce the number of cycles, reduce the template concentration, and/or increase denaturation time/temperature.
Template contained an exonuclease or was degraded Template may be sheared or contain exonuclease. Use a fresh template.
Primers contained impurities Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 μM of each primer.
Primers were designed or synthesized incorrectly by user or manufacturer Verify that primers have the correct sequence and are complementary to the template. Use a primer design program to avoid repetitive sequences, regions with high complementarity, etc. Perform a BLAST search to avoid primers that could amplify pseudogenes or that might prime unintended regions. Use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions) to calculate Tm. Use the lowest Tm of the primers.
Impure dNTPs were used Contaminants in the dNTP mix can lead to incomplete or incorrect amplification or PCR inhibition. Use high-quality dNTPs.
Water was impure Water could have been contaminated during prior pipetting events. Use fresh nuclease-free water.
 

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