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Setting up the laboratory to avoid contamination

General considerations

The ability of the Polymerase Chain Reaction to amplify a single molecule means that trace amounts of DNA contaminants could serve as templates, resulting in amplification of the wrong template (false positives). Consider the points mentioned below to avoid PCR contamination from sources such as:

  • Laboratory benches, equipment, and pipetting devices, which can be contaminated by previous DNA preparations, by plasmid DNA, or by purified restriction fragments.

  • Cross-contamination between samples.

  • Products from previous PCR amplifications.

Laboratory facilities

  • At a minimum, set up physically separated working places for:
      - Template preparation before PCR
      - Setting up PCR reactions
      - Post-PCR analysis.

  • Use thin-walled PCR tubes which are DNase and RNase free.

  • Use special aerosol-resistant pipette tips, and a dedicated (used only for PCR) set of pipettes, preferably positive displacement pipettes.

  • If possible, set up PCR reactions under a fume hood that is equipped with UV light. Under
    the fume hood, store a microcentrifuge and disposable gloves, which are used only for PCR.

Sample handling

  • Use sterile technique and always wear fresh gloves when working in the PCR area. Change gloves frequently, especially if you suspect they have become contaminated with solutions containing template DNA.

  • Always use new and/or sterilized glassware, plasticware, and pipettes to prepare PCR reagents and template DNA.

  • Autoclave all reagents and solutions that can be autoclaved without affecting their performance. Of course, primers, dNTPs and Taq DNA Polymerase should not be autoclaved.
    Have your own set of PCR reagents and solutions that are used only for PCR. Store these reagents in small aliquots.

  • When pipetting DNA, avoid creating aerosols that could carry contaminants.

  • Always include control reactions, for example a negative (“no DNA”) control which contains all reaction components except the template DNA, and a positive control that has been successfully used in previous PCRs.

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