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Troubleshooting

PCR Troubleshooting

Apresentação

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 Nonspecific Bands or Smeared Bands

Primer-Dimers

No Band or Faint Band Back to Top

Causes Related to Cycling Times and Temperatures

Too few Using too few PCR cycles can lead to insufficient amplification. Use 20–35 cycles. Use fewer cycles
cycles were when template concentration is high, and use more cycles when template concentration is low.
used

Extension If the extension time is too short, there will be insufficient time for complete replication of the target.
time was too Generally, use an extension time of 1 min/kb.
short

Annealing If the annealing time is too short, primers do not have enough time to bind to the template. Use an
time was too annealing time of at least 30 sec.
short

Annealing If the annealing temperature is too high, primers are unable to bind to the template. The rule of thumb is
temperature to use an annealing temperature that is 5°C lower than the Tm of the primer. To calculate the primer Tm,
was too high 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 If the denaturation temperature is too low, the DNA will not completely denature and amplification
temperature efficiency will be low. Use a denaturation temperature of 95°C.
was too low

Denaturation If the denaturation time is too long, DNA might be degraded. For the initial denaturation, use 3 min at
time was too 95°C; for denaturation during cycling, use 30 sec at 95°C.
long
Denaturation If the denaturation time is too short, the DNA will not completely denature and amplification efficiency will
time was too be low. For the initial denaturation, use 3 min to activate the polymerase; to denature the template
short during cycling, use 30 sec.
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Causes Related to PCR Components

dNTP If the dNTP concentration is too high, Mg2+ depletion occurs. Each dNTP should be present at 200 µM
concentration in the final reaction.
was too high

dNTP Each dNTP should be present at 200 µM in the final reaction.

concentration
was too low

PCR product GC-rich PCR products are difficult to amplify. To improve amplification, increase the annealing
has high GC temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient .
content DMSO or another secondary structure destabilizer can be added (do not exceed 10%).
(>65%)

Template was Template may be sheared or may contain PCR inhibitors. If inhibitors are suspected, dilute existing
damaged or template; otherwise, use fresh template and increase cycles. Try a control reaction in which you use a
degraded or pure plasmid with the addition of the template to determine if any inhibitory effects exist.
contained
inhibitors

Primers Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You
contained can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 µM of
impurities each primer.

Not enough Insufficient amplification can result if the initial amount of template is too low. Increase the number of
template was amplification cycles in increments of 5, or, if possible, increase the amount of template.
in the
reaction

Impure Contaminants in the dNTP mix can lead to incomplete or incorrect amplification or PCR inhibition. Use
dNTPs were high-quality dNTPs.
used

Primer Using an excessive concentration of primers can increase the chance of primers binding nonspecifically
concentration to undesired sites on the template or to each other. Use well-designed primers at 0.2–1 µM in the final
was too high reaction. In addition, verify that the correct concentration was supplied by the manufacturer.

Primer If the primer concentration is too low, annealing may be inefficient. Use well-designed primers at 0.2–1
concentration µM in the final reaction. In addition, verify that the correct concentration was supplied by the
was too low manufacturer.

Enzyme If the polymerase concentration is too low, not all PCR products will be fully replicated. The optimal
concentration enzyme concentration depends on the length and difficulty of the template.
was too low

Primers were Verify that primers have the correct sequence and are complementary to the template. Use a primer
designed or design program to avoid repetitive sequences, regions with high complementarity, etc. Perform a
synthesized BLAST search to avoid primers that could amplify pseudogenes or that might prime unintended
incorrectly by regions. Use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt
user or concentration and 0.2–1 µM primer (depending on your reaction conditions) to calculate Tm. Use the
manufacturer lowest Tm of the primers.

Target was PCR component concentrations and/or cycling conditions may not be sufficient for longer target
too long sequences. Reoptimize your existing assay protocol and/or increase the duration of PCR steps,
especially the extension step.

Water was Water could have been contaminated during prior pipetting events. Use fresh nuclease-free water.
impure

Not enough Insufficient or omitted magnesium will result in no or reduced PCR product. Use 1.5 mM in the final
Mg2+ 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 Use adequate units of enzyme. If you think the enzyme may be inactive, run a PCR with fresh
or inactive 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 Back to Top

Causes Related to Cycling Times and Temperatures

Too many Excessive cycling increases the opportunity for nonspecific amplification and errors. Use 20–35 cycles.
cycles were Use fewer cycles when template concentration is high, and use more cycles when template concentration
used is low.

Extension Excessive extension time can allow nonspecific amplification. Generally, use an extension time of 1
time was min/kb.
too long

Annealing Excessive annealing time may increase spurious priming. Use an annealing time of 30 sec.
time was
too long

Annealing If the annealing temperature is too low, primers may bind nonspecifically to the template. The rule of
temperature thumb is to use an annealing temperature that is 5°C lower than the Tm of the primer. To calculate the
was too low 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 If the ramp speed of the cycler is too slow, spurious annealing may occur due to lower temperature and
cycler sufficient time for nonspecific binding. If ramping speed is not set at the maximum speed for the cycler,
ramping increase to maximum ramp rate.
speed is
too slow

Calculated If the primer concentration is calculated incorrectly, the calculated annealing temperature will also be
primer Tm incorrect. To calculate the primer Tm, use the tool at
was www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 µM
inaccurate primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing
temperature.
Back to Top

Causes Related to PCR Components

Primers Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You
contain can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 µM of
impurities each primer.

Too much Using a high concentration of primers may increase the chance of primers binding to nonspecific sites
primer was on the template or to each other. Use well-designed primers at 0.2–1 µM in the final reaction. In
added addition, verify that the correct concentration was supplied by the manufacturer.

Primers were Verify that primers have the correct sequence and are complementary to the template. Use a primer
designed or design program to avoid repetitive sequences, regions with high complementarity, etc. Perform a
synthesized BLAST search to avoid primers that could amplify pseudogenes or that might prime unintended regions.
incorrectly Use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration
by user or and 0.2–1 µM primer (depending on your reaction conditions) to calculate Tm. Use the lowest Tm of the
manufacturer primers.

Impure Contaminants in the dNTP mix can lead to incomplete or incorrect amplification or PCR inhibition. Use
dNTPs were high-quality dNTPs.
used

Too much Using high concentrations of magnesium increases the likelihood of nonspecific primer binding and
Mg2+ was unwanted product formation. Reduce the amount of magnesium in the final reaction.
added

Impure water Water could have been contaminated during prior pipetting events. Use fresh nuclease-free water.
was used

Smeared Bands Back to Top

 Causes Related to Cycling Times and Temperatures

Too many Excessive cycling increases the opportunity for nonspecific amplification and errors. Use 20–35 cycles.
cycles were Use fewer cycles when template concentration is high, and use more cycles when template concentration
used is low.

Extension Excessive extension time can allow nonspecific amplification. Generally, use an extension time of 1
time was min/kb.
too long

Annealing Excessive annealing time may increase spurious priming. Use an annealing time of 30 sec.
time was
too long

Annealing If the annealing temperature is too low, primers may bind nonspecifically to the template. The rule of
temperature thumb is to use an annealing temperature that is 5°C lower than the Tm of the primer. To calculate the
was too low 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 If the ramp speed of the cycler is too slow, spurious annealing may occur due to lower temperature and
cycler sufficient time for nonspecific binding. If ramping speed is not set at the maximum speed for the cycler,
ramping increase to maximum ramp rate.
speed is
too slow

Calculated If the primer concentration is calculated incorrectly, the calculated annealing temperature will also be
primer Tm incorrect. To calculate the primer Tm, use the tool at
was www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 µM
inaccurate primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing
temperature.
Back to Top

Causes Related to PCR Components

Too much If the template concentration is too high, the polymerase can be inhibited due to carryover of inhibitors
template was or inefficient denaturation. Reduce the number of cycles, reduce the template concentration, and/or
added increase denaturation time/temperature.

Template Template may be sheared or contain exonuclease. Use a fresh template.

contained an
exonuclease
or was
degraded

Primers Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You
contained can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 µM of
impurities each primer.

Primers were Verify that primers have the correct sequence and are complementary to the template. Use a primer
designed or design program to avoid repetitive sequences, regions with high complementarity, etc. Perform a
synthesized BLAST search to avoid primers that could amplify pseudogenes or that might prime unintended regions.
incorrectly Use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration
by user or and 0.2–1 µM primer (depending on your reaction conditions) to calculate Tm. Use the lowest Tm of the
manufacturer primers.

Impure Contaminants in the dNTP mix can lead to incomplete or incorrect amplification or PCR inhibition. Use
dNTPs were high-quality dNTPs.
used

Water was Water could have been contaminated during prior pipetting events. Use fresh nuclease-free water.
impure
Back to Top

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