PCR and qPCR
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Could your PCR be affected by contamination?

Follow these tips to help eliminate false amplification.

It is frustrating when your eagerly anticipated PCR data show unexpected amplicons. You know your primers are correct, you are certain of the amplicon length, and you definitely used the correct template. So what could be wrong? There can be various sources of contamination during PCR, leading to myriad observations that may require troubleshooting. A common observation is excessive or unexpected signal, typically caused by contamination of reagents with template, genomic DNA, or amplicons from previous reactions.

Below, we provide some tips to help prevent false amplification due to contamination.

Protect reaction setup from genomic DNA:

  • Use DNase to degrade genomic DNA before performing reverse transcription. If the aim of your experiment is to measure RNA expression, treat your RNA sample with DNase, and then heat inactivate the DNase before performing reverse transcription.
  • Design your assays to span exon junctions. To minimize the risk of amplifying contaminating genomic DNA, whenever possible, ensure that your primers span 2 exons (Figure 1).

    Figure 1. Primer design across exon boundaries. To avoid amplifying genomic DNA, design primers with complementarity to the ends of adjacent exons.

  • Always incorporate a “no-RT” (–RT) control in your experiment. A –RT control is created by omitting reverse transcriptase in the reverse transcription step and helps to identify genomic DNA contamination in an RNA preparation. Perform this control for any assay that might amplify genomic DNA.

Reduce the risk of cross-contamination from the amplicon/template and previous reactions:

  • Use a unidirectional workflow during experiment setup. Separating pre- and post-amplification areas is key to preventing contamination. Prepare your PCR master mix in a template-free room (see next bullet), using reagents that never come into contact with potential sources of contamination. Maintain a separate area for analyzing PCR amplicons.
  • Maintain a clean room for preparing master mixes. Prepare your PCR master mix in one room, and then add your template in a separate room to avoid introducing template into the clean room. Keep enzyme mixes, water, primers and probes, pipettes, tubes, tips, and plates in a room where template is not isolated or stored.
  • Regularly decontaminate equipment. Clean pipettes and other non-porous surfaces with 5% bleach solution to degrade any DNA that may be present. It is easiest to use a spray bottle for this purpose. Leave the solution on the surface for a few minutes to ensure complete degradation of DNA. Alternatively, you can use UV sterilization to decontaminate equipment, including tubes, racks, and pipettes.
  • Use positive displacement or filter tips for reaction setup. Pipettes are a common source of contamination by aerosols. Filter tips provide a barrier between the pipettes and the liquid being measured, preventing the transfer of aerosols into samples and reagents. Positive displacement pipettes have no air interface between the piston and reagents being measured and, therefore, limit the risk of aerosol contamination.

Exercise general good laboratory practice:

  • Wear a clean lab coat and clean gloves when preparing samples. Contaminating DNA that may be present on lab coats and used gloves is easily amplified during PCR. Use a clean lab coat and change your gloves frequently to reduce the risk of transferring contaminants to your reaction setup.
  • Store oligonucleotides in aliquots. We have found that multiple freeze-thaw cycles do not adversely affect the quality of our PCR oligo stock solutions. However, to minimize the risk of contaminating stock solutions, store your oligos at –20°C in single-experiment aliquots. Storing your oligos in aliquots also allows you to repeat the experiment with fresh material if contamination is detected in an experiment using those oligos.

If unexpected amplification in your PCR is confirmed to be due to contamination:

  • Replace all reagents and stock buffers. Discard all existing reagents and repeat the experiment with fresh stocks.

Product focus

Assays, probes, and controls for qPCR and PCR

PrimeTime® qPCR Assays
  • 5′ nuclease, probe-based assays—the gold standard for quantitative gene expression studies
  • Primer-based assays—designed for intercalating dye experiments

Create custom assays that are designed using our proprietary bioinformatics algorithms for any target and to your specific parameters. Alternatively, select one of our predesigned assays for human, mouse, and rat mRNA targets that are supported by our bioinformatics algorithms and up-to-date sequence information.

Learn more at www.idtdna.com/PrimeTime. For assistance with assay design, contact our scientific application specialists at applicationsupport@idtdna.com.

Double-Quenched Probes

ZEN™ or TAO™ Double-Quenched Probes have a 5′ fluorophore, an internal quencher (ZEN or TAO quencher), and Iowa Black FQ as the 3′ quencher. These probes provide consistently earlier Cq values and improved precision, when compared to traditional, single-quenched qPCR probes.

Learn more at www.idtdna.com/qPCRprobes.

gBlocks® Gene Fragments

gBlocks Gene Fragments are double-stranded, 125–2000 bp DNA molecules. They are ideal for use as qPCR controls and standards, as well as for gene construction and editing applications. These affordable gene fragments are sequence-verified, ship in a few working days, and save laboratory time.

Learn more at www.idtdna.com/gBlocks.

 

Free tools for qPCR and PCR assay design

Explore IDT SciTools® Web Tools for free, online tools for qPCR probe design and analysis. The design engines for these tools use sophisticated formulas that, for example, take into account nearest neighbor analysis to calculate Tm, and provide the very best qPCR assay designs.


Additional reading

Discover more resources for qPCR and PCR assay design:

Author: Nicola Brookman-Amissah, PhD, is the manager of scientific communications at IDT.

© 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.


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