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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 commercial clean up kit
    • 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 trademarke of New England Biolabs, inc.