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PCR Troubleshooting Guide

The following guide can be used to troubleshoot PCR reactions. Use our Tm calculator to help plan experiments and click here for optimization tips.

Observation Possible Cause Solution
Sequence Errors
 Low fidelity polymerase
  • Choose a higher fidelity polymerase such as Q5® (NEB #M0491), Phusion™ (NEB #M0530) DNA Polymerases
Suboptimal reaction conditions
  • Reduce number of cycles
  • Decrease extension time
  • Decrease Mg++ concentration in the reaction
Unbalanced nucleotide concentrations
  • Prepare fresh deoxynucleotide mixes
Template DNA has been damaged
  • Start with a fresh template
  • Try repairing DNA template with the PreCR® Repair Mix (NEB #M0309)
  • Limit UV exposure time when analyzing or excising PCR product from the gel
Desired sequence may be toxic to host
  • Clone into a non-expression vector
  • Use a low-copy number cloning vector
Incorrect Product Size
 Incorrect annealing temperature
Mispriming
  • Verify that primers have no additional complementary regions within the template DNA
Improper Mg++ concentration
  • Adjust Mg++ concentration in 0.2–1 mM increments
Nuclease contamination
  • Repeat reactions using fresh solutions
No Product
 Incorrect annealing temperature
  • Recalculate primer Tm values using the NEB Tm calculator
  • Test an annealing temperature gradient, starting at 5°C below the lower Tm of the primer pair
Poor primer design
  • Check specific product literature for recommended primer design
  • Verify that primers are non-complementary, both internally and to each other
  • Increase length of primer
Poor primer specificity
  • Verify that oligos are complementary to proper target sequence
Insufficient primer concentration
  • Primer concentration can range from 0.05–1 µM in the reaction. Please see specific product literature for ideal conditions
Missing reaction component
  • Repeat reaction setup
Suboptimal reaction conditions
  • Optimize Mg++ concentration by testing 0.2–1 mM increments
  • Thoroughly mix Mg++ solution and buffer prior to adding to the reaction
  • Optimize annealing temperature by testing an annealing temperature gradient, starting at 5°C below the lower Tm of the primer pair
Poor template quality
  • Analyze DNA via gel electrophoresis before and after incubation with Mg++
  • Check 260/280 ratio of DNA template
Presence of inhibitor in reaction
  • Further purify starting template by alcohol precipitation, drop dialysis or use Monarch® Spin PCR & DNA Cleanup Kit (5 μg) (NEB #T1130)
  • Decrease sample volume
Insufficient number of cycles
  • Rerun the reaction with more cycles
Incorrect thermocycler programming
  • Check program, verify times and temperatures
Inconsistent block temperature
  • Test calibration of heating block
Contamination of reaction tubes
or solutions
  • Autoclave empty reaction tubes prior to use to eliminate biological inhibitors
  • Prepare fresh solutions or use new reagents and new tubes
Complex template
  • Use Q5 High-Fidelity (NEB #M0491) or OneTaq® DNA Polymerases (NEB #M0480)
  • For GC-rich templates, use Q5 High-Fidelity (NEB #M0491) or OneTaq® DNA Polymerases. Include the appropriate GC enhancer.
  • For longer templates, we recommend LongAmp® Taq DNA Polymerase or Q5 high-Fidelity DNA polymerase or Q5 Hot-Start High-Fidelity DNA Polymerase (NEB #M0493)
Multiple or Non-Specific Products
 Premature replication
  • Use a hot start polymerase, such as OneTaq Hot Start DNA Polymerase
  • Set up reactions on ice using chilled components and add samples to thermocycler preheated to the denaturation temperature
Primer annealing temperature too low
  • Increase annealing temperature
Incorrect Mg++ concentration
  • Adjust Mg++ in 0.2–1 mM increments
Poor primer design
  • Check specific product literature for recommended primer design
  • Verify that primers are non-complementary, both internally and to each other
  • Increase length of primer
  • Avoid GC-rich 3´ ends
Excess primer
  • Primer concentration can range from 0.05–1 µM in the reaction. Please see specific product literature for ideal conditions.
Contamination with exogenous DNA
  • Use positive displacement pipettes or non-aerosol tips
  • Set-up dedicated work area and pipettor for reaction setup
  • Wear gloves during reaction setup
Incorrect template concentration
  • For low complexity templates (i.e. plasmid, lambda, BAC DNA), use 1 pg–10 ng of DNA per 50 µl reaction
  • For higher complexity templates (i.e. genomic DNA), use 1 ng–1 µg of DNA per 50 µl reaction

Phusion DNA Polymerase was developed by Finnzymes Oy, now a part of Thermo Fisher Scientific. This product is manufactured by New England Biolabs, Inc. under agreement with, and under the performance specifications of Thermo Fisher Scientific.
PHUSION™ is a registered trademark of Thermo Fisher Scientific.
Q5® is a trademark of New England Biolabs, inc.