AAT Bioquest

How can I improve PCR efficiency?

Posted March 25, 2022


Contamination is the main reason for lower PCR efficiency. Trace amounts of DNA contaminants can serve as templates, resulting in false positives by amplification of the wrong template. The key to improving PCR efficiency is to avoid PCR contamination while setting up the laboratory and also while handling the samples.

How to set up the laboratory to avoid cross-contamination of samples and improve PCR efficiency:

  • Use three separated working places for preparing the template before PCR, for setting up PCR reactions, and for post-PCR analysis
  • Use a dedicated set of pipettes only for PCR, preferably positive displacement pipettes
  • Use special pipette tips that are aerosol-resistant
  • Use thin-walled PCR tubes that are free of DNase and RNase
  • Set up PCR reactions under a fume hood equipped with a UV light
  • Scrupulously clean all equipment, pipetted devices, working surfaces and laboratory benches to remove all traces of contamination by previous DNA preparations, purified restriction fragments, or plasmid DNA. 

How to handle samples to avoid cross-contamination and improve efficiency of PCR:

  • Always prepare PCR reagents and template DNA using new and/or properly sterilized pipettes, glassware, and plastic ware
  • Always wear a fresh pair of gloves before handling any samples or equipment when working in the PCR area
  • Change gloves often during the assay and especially if you suspect they may have become contaminated with any solution or container containing template DNA
  • When pipetting DNA, avoid creating aerosols that can carry contaminants
  • Keep your own set of PCR reagents and solutions and use them only for PCR
  • Autoclave all reagents and solutions that can be autoclaved without having an adverse effect on their performance – this excludes primers, Taq Polymerase and dNTPs, which should not be autoclaved

Other ways to improve efficiency besides maintaining purity is specificity with fluorescent DNA-binding dyes, incorporating an internal standard control such as a second primer, and optimizing primer and probe concentrations.

Additional resources

Strategies to Improve Efficiency and Specificity of Degenerate Primers in PCR

dTTP *100 mM PCR Grade*