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  • Taq PCR Kit Troubleshooting Guide

    The following guide can be used to troubleshoot PCR reactions using Taq DNA Polymerase. We also provide optimization tips for PCR with Taq DNA Polymerase.

    Observation Possible Cause(s) Solution(s)
    No amplification product Poor 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
    • Increase primer concentration, ensuring it is in the range of  0.05–1.0 μM.
    Missing reaction component
    • Repeat reaction setup.
    Target sequence not present in template DNA
    • Try other sources of template DNA.
    Poor reaction conditions
    • Optimize (Mg++), annealing temperature and extension time.
    • Thoroughly mix Mg++ solution.
    • Check primer concentrations.
    • Recalculate primer Tms using the NEB Tm Calculator.
    Questionable template quality
    • Analyze DNA via gel electrophoresis after incubation with Mg++.
    Inhibitory substance in reaction
    • Decrease sample volume, added to the reaction
    • Try alcohol precipitation or drop dialysis to further purify DNA.
    Insufficient number of cycles
    • Return reaction to thermocycler and run additional cycles.
    Incorrect thermocycler programming
    • Check program, verify times and temperatures.
    Inconsistent block temperature
    • Test calibration of heating block.
    Reaction tubes or solutions contaminated
    • Autoclave tubes prior to use to eliminate biological inhibitors.
    Multiple or non-specific products Premature Taq DNA
    Polymerase replication
    • Set up reactions on ice with chilled components. Add samples to pre-heated (95°C) thermocycler.
    Primer annealing temperature too low
    • Use a Hot Start Taq DNA Polymerase (NEB #M0490)
    • Recalculate primer Tms using the NEB Tm Calculator.
    • Raise annealing temperature in 2°C increments.
    Insufficient mixing of reaction buffer
    • Reaction buffer must be thoroughly mixed.
    Improper Mg++ concentration
    • Adjust Mg++ concentration in 0.5 mM increments.
    Poor primer design
    • Verify that primers have no complementary regions – either internally or to each other.
    • Try longer primers.
    • Avoid GC-rich 3´ ends.
    Excess primer
    • Reduce primer concentration, ensuring that it falls in the range of 0.05–1.0 µM.
    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.
    Multiple target sequences in template DNA
    • Redesign primers with higher specificity to target sequence.
    Clones contain mutations Excessive Mg++
    • Use minimal concentration of Mg++ to produce desired amount of product.
    Wild-type target sequence may be toxic to host
    • Clone into non-expression vector.
    • Use low-copy cloning vector.