The goal of (classic) PCR is the generation of a reliable and reproducible result. For certain applications, the yield of the PCR product is also relevant. For these reactions, care must be taken to ensure that samples are not compromised and that the PCR workflow remains stable. Specifically, this translates to minimizing the introduction of contaminations that could lead to false positive or false negative results or even inhibit the PCR reaction. Furthermore, the reaction conditions should be as identical as possible for each individual sample within a run and also be transferable to subsequent reactions (of the same method). This refers to the composition of the reactions as well as to the type of temperature control in the cycler. User errors, of course, are to be avoided as much as possible.
Below, we will demonstrate the challenges which are encountered during preparation and throughout the run of a PCR – and the approaches to solutions which exist with respect to instruments and consumables used for the standardization of the PCR workflows.
The dispensing of reaction components into PCR-vessels, or plates, respectively, comprises multiple challenges that must be overcome:
The exact and precise dosing of the individual components is indispensable when aiming for the most identical reaction conditions possible. In addition to a good pipetting technique, selecting the right tool is critical. PCR master-mixes frequently contain substances which increase viscosity or generate foam. During the pipetting process, these lead to substantial wetting of the pipette tips, thus reducing pipetting accuracy. The use of direct dispensing systems (figure 1) or alternative pipette tips that are less prone to wetting (figure 2) can improve the accuracy and precision of the pipetting process. (Pipetting PCR mixtures)
If a large number of PCRs are performed, it is also worth considering automation of the setup. (Automated PCR Set-up)