Best Practices for Reliable PCR Results

Few failures in molecular biology are as invisible and as damaging as contamination. PCR doesn't just amplify your target sequence – it amplifies any stray DNA or RNA that makes it into the reaction. Anyone who has seen a signal in a no-template control knows exactly what that means: an entire run, potentially wasted.

This post covers how DNA and RNA contamination occurs, how to recognise it when it happens, and what practical steps keep your PCR results reliable over the long term.

Where Contamination Comes From – and Why It's Hard to Shake

The sensitivity that makes PCR so powerful is also what makes it unforgiving. A few stray DNA copies are all it takes for a false positive. Contamination in a PCR lab tends to fall into three categories.

Amplicon carryover is the most common culprit. Amplification products from previous runs settle as aerosols on surfaces, pipettes, and reagents. Once distributed through the air of a lab, they are nearly impossible to eliminate entirely.

Cross-sample contamination occurs when DNA or RNA from one sample transfers to another – through pipetting errors, tips without filters, or careless handling during sample preparation.

Environmental contamination covers anything that enters the reaction from lab air, surfaces, equipment, skin, or breath. RNA work carries an additional challenge: RNases are ubiquitous and actively degrade target sequences, even in trace amounts.

Detecting Contamination: Controls Are Non-Negotiable

Contamination is invisible to the naked eye. The only reliable way to catch it is through well-designed controls that run alongside every PCR experiment.

The no-template control (NTC) contains all reagents but no target DNA or RNA. A signal in the NTC points directly to contamination in a reagent, the nuclease-free water, or on the work surface.

The extraction control follows the full sample preparation process from the beginning. It contains nucleic-acid-free material and flags contamination introduced during the extraction step itself, not just during PCR setup.

Running reactions in triplicate matters more than it might seem. Single-replicate results at low signal levels leave no room to distinguish a genuinely low-positive sample from a contamination artefact. Multiple determinations are especially important when signals fall near the detection threshold.

Prevention: Spatial Separation Is the Foundation

No decontamination spray compensates for a disorganised lab. The single most effective contamination control measure is physical separation of pre- and post-amplification areas.

The pre-PCR area is where master mixes are prepared and samples are added. Pipettes, reagents, consumables, and personal protective equipment kept in this area must never come into contact with amplification products. Those belong exclusively in the post-PCR area. Anyone returning from there changes gloves and lab coat before re-entering the pre-PCR zone.

In practice this means dedicated pipettes for each area, separate refrigerators for kit storage and sample storage, and a one-way workflow that everyone on the team understands and follows without exception.

Decontaminating Surfaces and Equipment

Standard alcohols do not destroy nucleic acids. Ethanol disinfects, but it doesn't denature DNA – which is why 70% ethanol alone is insufficient as a surface treatment in a PCR lab.

Two approaches have proven effective for surface decontamination: freshly prepared sodium hypochlorite solution (10–15%, made daily) and specialist decontamination reagents that eliminate DNA, RNA, DNases, and RNases in a single step. UV irradiation in a PCR workstation adds an extra layer of protection, but it doesn't replace chemical decontamination.

Centrifuges and vortexers tend to be overlooked. Aerosol generation during centrifugation makes them a common source of contamination. Wiping down the interior surfaces regularly should be part of the lab routine.

Consumables: Quality Has a Real Impact

The quality of consumables is not a minor detail. Tips without aerosol filters offer no protection against carryover into the pipette barrel. Tubes that aren't certified DNase- and RNase-free introduce background risk from the start.

Eppendorf products available at häberle LABORTECHNIK include a full range of PCR-clean consumables – from ep Dualfilter T.I.P.S. to Biopur-grade PCR tubes and the Eppendorf PCR-Cooler, which keeps reactions at 0°C during setup without an ice bath or any risk of ice melt contamination. All materials are certified free of human DNA, DNase, RNase, and PCR inhibitors.

BRAND consumables, also stocked at häberle LABORTECHNIK, are produced in cleanroom conditions and are certified free of DNA, RNA, DNase, and RNase. For sensitive molecular biology work, this certification is not a marketing claim – it's a minimum requirement.

RNA Work Demands Extra Caution

RNA is less stable than DNA, and RNases are nearly everywhere – on skin, in aerosols, on lab equipment. A reaction tube briefly touched with bare hands can compromise an entire RT-PCR setup.

Wearing disposable gloves at all times, working with nuclease-free water in single-use aliquots, and setting up reactions on freshly decontaminated surfaces aren't optional precautions in RNA analysis. They're the baseline.

Summary

Reliable PCR results don't happen by luck. They come from a well-organised lab, properly maintained equipment, certified consumables, and controls that run in every experiment without exception.

Maintaining spatial separation between pre- and post-amplification areas, decontaminating surfaces with the right reagents, and consistently using filter tips and PCR-clean tubes significantly reduces contamination risk – and removes the guesswork from interpreting results.

häberle LABORTECHNIK stocks PCR consumables from Eppendorf and BRAND that meet these standards. The team is happy to help you find the right products for your workflow.

Explore PCR consumables at häberle LABORTECHNIK →