Bio-Rad's HardShell 96-Well PCR Plate
by Jennifer Placek, Gene Expression Specialist, Bio-Rad Laboratories, and Angelica Olcott, Gene Expression Specialist, Bio-Rad Laboratories
Conventional PCR, qPCR, digital PCR and NGS applications all use PCR plastics to facilitate sample preparation and amplification steps. As these technologies have expanded their utility, the demand for high-performance plastics has also increased. In today’s budget constrained environments, purchasing lower cost standard PCR plastics can appear to be a useful solution. However, researchers should be aware that the lower cost plastics often associate with inferior quality and thus may display lower heat stability, which could have adverse effects on amplification reactions, ultimately impacting results.
The choice of PCR plastics has a substantial impact in a researcher’s assay that they may not be aware of initially, but it really can help ensure more optimal results. Thus, it is especially important to consider the necessary properties of PCR plates before running an assay of precious samples, whether you are in a research or clinical laboratory.
Does the choice of PCR plate meet the requirements of the thermal cycler?
Understanding the requirements and design of the chosen thermal cycler is the first step towards selecting an appropriate PCR plate. For example, is the PCR plate height suitable for the instrument? Identifying the thermal cycler’s lid mechanism (i.e., a fixed-height or adjustable lid) can aid in correctly determining the plate specifications. An instrument with an adjustable lid can utilize high-profile (20.7mm high) or low-profile (15.5mm high) standard PCR plates, while one with a fixed-height lid can only use one type of plate profile.
The lid of a thermal cycler contains a heated plate assembly that applies a specific amount of pressure to the plate caps or seal when the lid is closed—failing to use the required plate dimensions will result in the incorrect application of pressure. Using a plate that is too tall will cause the wells to compress during thermal cycling as too much pressure is applied, whereas using a plate that is too small will result in sample condensation or evaporation as insufficient pressure is applied.
Another key consideration is whether the plate skirt will be compatible with the thermal cycler. Providing rigidity, the skirt is the outer frame of the plate, and comes in three options: full-skirted, semi-skirted, or unskirted. The correct skirt length will depend on the instrument’s thermal block design; for example, if the thermal block is encased in the deck of the instrument, an unskirted plate is the only compatible option. Additionally, if the PCR plate will be used with an automated liquid handler, then a semi- or full-skirted plate is essential to allow the robot to securely grip the plate.
What is the best color?
The color of a PCR plate’s wells directly affects real-time PCR results. Researchers must therefore determine if their plate wells should be clear or white—a decision that often relates to whether signal background is more important than visual confirmation of samples in the plate wells.
For example, clear well plates allow the user to visually monitor pipetting from the side, top or bottom of the plate, yet they also have a higher chance of fluorescent signal bleeding into a neighboring well and artificially increasing the signal. Bleed-through is less of an issue with white wells, and they also offer a better signal-to-noise ratio, which can be important for amplifying and detecting a signal from low concentration samples. However, discerning liquid levels in a white well or colored well plate is only possible by directly looking down at the plate well.
High quality plate design and manufacture
Researchers should avoid PCR plates that easily twist and bend, for example, plates manufactured using a one-component design (i.e., made entirely of polypropylene resin using a single mold). Not only are they challenging to handle, but they may not sit flat in a thermal cycler block, resulting in the uneven heating of samples or sample evaporation, which can compromise the amplification process. Also, these flexible plates are not compatible with automation.
Two-component plates (as pictured above) prevent these problems by using a rigid polycarbonate frame as well as polypropylene wells, enabling the plate to retain its dimensions throughout the thermal cycling process, keeping the plate sealed, and providing a sturdy surface for robotic plate grippers to make contact.
Beyond the manufacturing design, researchers should obtain plates from a trusted manufacturer that can ensure consistent, high-quality standards. Low quality PCR plates can be prone to heat transfer that is less uniform, and the resulting temperature instability during the thermal cycling process can lead to incomplete or uneven DNA amplification. A reliable manufacturer would monitor the in-process and final product stages to guarantee consistent plate dimensions, low background fluorescence, and that the product is defect-free. In addition, PCR plate batches should be certified that they are free of contaminants such as DNAse, RNAse, and human DNA.
Having chosen their PCR plates, researchers should not forget to pair high quality plastics with high performance qPCR and PCR instrumentation. Higher quality instrument systems provide another level of assurance when performing the preparative steps involved in sophisticated molecular techniques such as digital PCR and RNA sequencing.
Understanding how the PCR plate can impact assays underpins the selection of plates for a specific application. PCR plate compatibility can be based on product quality and design, as well as instrumentation and throughput. Luckily, PCR plate guides—like the PCR Plastic Selector—are available to help researchers identify which plates are compatible for specific instruments.
Determining the optimal PCR plate options gives researchers the confidence that the plates chosen for their conventional PCR, qPCR, digital PCR and NGS assays will perform as expected.