3 Recommendations to Keep in Mind When Performing PCR

Published: 28/08/26 14:05 Categories: Microbiology

Obtaining reliable Real-Time PCR results does not depend solely on the quality of the reagents or equipment used; proper sample and reaction preparation is essential to ensure the success of the assay.
Small details during steps such as DNA extraction, reaction mix preparation, or sample handling can make a significant difference in the sensitivity, reproducibility, and accuracy of the results.
Below, we share some practical recommendations to help you optimize your workflow and improve the performance of your analyses.

 

1. Choose a DNA Extraction Kit Suitable for Your Matrix

This is a key step in obtaining high-quality DNA and ensuring the assay performs correctly. Depending on the type of matrix — for example, high-fat foods, environmental samples, or highly contaminated water — different substances that interfere with amplification may be present. The presence of polymerase inhibitors, such as polysaccharides, phenolic compounds, or salts, can reduce PCR efficiency and lead to weak or inconsistent results, or even false negatives.

Therefore, it is important to select an extraction kit that has been specifically validated or recommended for the matrix of interest.





2. Prepare a Reaction Mix Master Mix

When preparing the reaction mix, we recommend preparing a pool containing the volume required for all the samples to be analyzed, while also allowing for a small excess to compensate for pipetting losses. Preparing a pool helps reduce pipetting error, as the reaction components are distributed homogeneously before being added to each sample.

It also helps streamline the process and maintain more consistent conditions across all reactions. It is advisable to keep the mix at the temperature conditions specified by the manufacturer and avoid prolonged exposure to room temperature, especially when working with sensitive enzymes.

3. Remove Bubbles Before Placing the Tubes in the Thermocycler

Before placing the tubes or plate in the thermocycler, it is important to check that there are no bubbles in the reaction mix. Bubbles can alter the optical contact between the sample and the detection system and may interfere with the accurate reading of the signal. To prevent this, once the reaction has been prepared, we recommend visually checking the tubes and performing a brief centrifugation to bring the entire volume to the bottom of the tube and remove any potential bubbles. This simple step helps improve the uniformity of fluorescence readings and, consequently, the reproducibility and reliability of the results.

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