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PCR troubleshooting guide

In molecular cloning, after the synthesis of cDNA from mRNA molecule templates, a PCR program must be designed to amplify the gene of interest, as well as add additional elements such as restriction sites or detection/purification tags. Intrinsic properties of gene sequences such as high GC content, long stretches of the same polynucleotide, and sequences encoding hairpin loop structures can all hinder PCR efficiency.

Use the troubleshooting guide below to identify the cause of PCR failure and improve PCR efficiency or get your desired gene in the vector you want the easy way with GenEZ™ ORF clones. Start with a search for your gene.


Problem Causes Solutions
Low or no PCR product yield
Number of PCR cycles is insufficient
  • Increase number of PCR cycles by 5.
Template is degraded
  • Use electrophoresis to check DNA quality.
Template is contaminated with PC inhibitors
  • Check DNA ratio of absorbance at 260 and 280 nm. Pure DNA should have a 260/280 ratio of ≥ 1.8.
  • Use less volume of the template in the reaction.
  • Use DNA clean-up kit or ethanol precipitation to remove contaminants.
Thermocycler program annealing and extension temperatures are not optimal
  • Follow general rules of PCR design: Annealing temperature = lowest primer Tm - 5 °C, Extension temperature = 72 °C.
  • Decrease annealing temperature by 6 to 10°C in stepwise fashion.
Reaction is missing Taq polymerase or other reaction component
  • Make sure each component was added to PCR reaction.
Primer concentration too low
  • Check primer concentration; increase concentration if necessary.
Target sequence is not in DNA template
  • Re-extract DNA from source.
  • Test another region of template.
Not enough template
  • Increase concentration of DNA template.
Reaction component concentrations not optimal
  • Check recommended primer concentrations (normally from 0.05–1 mM) and Mg++ concentrations (normally from 0.2–1 mM).
Reaction mix components are compromised
  • Check expiration date of components.
  • Aliquot biological components of reaction mixture and avoid multiple freeze-thaw cycles.
Primer design not optimal (causing non-specific annealing, or primer dimer formation)
  • Follow general rules of primer design: Length from 18-30 nucleotides, GC content from 40-60%, Tm of primers within 5°C of each other.
  • Avoid stretches of 4 or more of the same nucleotide or dinucleotide repeats.
  • Avoid self-complementary sequences within primers.
Multiple/non-specific products from PCR reaction
Primer design not optimal (causing non-specific annealing, or primer dimer formation)
  • Follow general rules of primer design: Length from 18-30 nucleotides, GC content from 40-60%, Tm of primers within 5°C of each other.
  • Avoid stretches of 4 or more of the same nucleotide or dinucleotide repeats.
  • Avoid self-complementary sequences within primers.
Template or reaction mixture components are contaminated
  • Re-extract template.
  • Try new reaction mixture.
  • Use filter pipette tips and wear gloves during reaction set-up.
Annealing temperature too low
  • Incrementally increase annealing temperature.
Primer concentration too high
  • Use less primer.
Template concentration is not optimal
  • For plasmids use 1 pg–10 ng of DNA / 50 µl reaction.
  • For genomic DNA use 1 ng–1 µg of DNA / 50 µl reaction.

PCR protocol »
 
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