Troubleshooting

PCR
PCR is a three-step, cyclic process, where new
strands of DNA are created through successive
rounds of denaturation, annealing, and
extension using the enzyme DNA polymerase.
PCR can fail for various reasons, in part due
to its sensitivity to contamination causing
amplification of spurious DNA products.

A DNA Template that contains the target sequence.

DNA Polymerase (Taq) this is the enzyme that
synthesizes new DNA.
Nucleotides (dATP, dTTP, dGTP, dCTP) the building

Components of
blocks of DNA.
Primers these are short DNS fragments that are
a Successful PCR complimentary to the target sequence of interest.
Buffers buffers containing magnesium ions provide
the required conditions for the polymerase to work.

Expert Tip:
Prepare the master mix in only one tube to prevent pipetting variations and use a tube large enough
to hold the entire volume of the master mix.
Alliqout immediately afterwards to avoid multiple thawing that can negatively impact PCR reproducibility.

Missing Components: Check that you

included all required components and retry.
Denaturing Temperature Too Low:
If temperature is too low, denaturation will
not be complete and amplification will be low.
Troubleshooting A denaturation temperature of 95 C is recommended.

No Bands Annealing Temperature Too High:

If temperature is too high, primers will not bind to the
template. Use a temperature that is 5 C lower than
the Tm of the primer.
Too Few PCR Cycles: Use 20-25 cycles
(fewer if template concentration is high and more
if concentration is low).
Bad Template: Template could be sheared or
contain PCR inhibitors; dilute existing template or use
fresh template and increase cycles.
Contamination: Contaminants in the primers,
dNTPs, or water can inhibit PCR. Use only the best
quality reagents and nuclease-free water.

Expert Tip, In The Event of No or Low Amplification:

Optimize denaturation and/or annealing temperature with a gradient.
Use PCR enhancers such as DMSO or BSA. Each require empirical testing for the specific
combination of template and primer

Too Many Cycles: Excessive cycling can result

in non-specific amplification and errors. Use 20-35
cycles and fewer when template concentration is high.
Annealing Temperature Too Low: Under
lower temperatures, primers may bind nonspecifically
Troubleshooting to the template. Try increasing your annealing
temperature.
Non-specific Bands Annealing Time Too Long: Longer annealing
times can result in spurious priming. Keep annealing
time to 30 sec.
Extension Time Too Long: Excessive
extension can cause nonspecific amplification. Keep
extension time to 60 seconds/kb.
Heating Too Slow: If ramping speed is too slow,
spurious annealing may occur. Set your ramping speed
to maximum.
Contamination: Contaminants in your
primers or dNTPs can lead to incomplete or incorrect
amplification. Use only the highest quality reagents.
Too Much Mg++: Excessive magnesium
increases the likelihood of nonspecific primer binding.
Reduce the amount of Mg++ in your final reaction.

Expert Tip, In Case of Non-specific Amplifications:

Use hot-start protocols. Make sure your cycler is properly calibrated with rapid temperature ramp-up.
For new primers, run optimization with a single primer to determine non-specificity.
Titrate Mg++ to optimize the concentration for your PCR reaction.

Annealing or Extension Times Too Long:

Annealing times of 30 seconds and extension times
of 1 min/kb are ideal.
Temperature Too Low or Thermal
Cycler Ramping Slowly:
Troubleshooting Low annealing temperature may cause primers to bind
non-specifically. Increase your temperature and, for
Smeared Bands greater accuracy, optimize using a thermal gradient.
Too Much Template Was Added:
If template concentration is too high, polymerase
can be inhibited. Reduce the number of cycles, reduce
template concentration, and increase denaturation
time and/or temperature.
Degraded Template: If template is degraded,
use a fresh sample.
Template Contained An Exonuclease:
If this is suspected, use a fresh sample.
Contamination: Contamination of primers,
dNTPs, or impure water can result in incomplete or
incorrect amplificationuse high quality reagents and
nuclease-free water.
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