Jan. 06, 2025
There are many thousands of PCR plastic consumables (plates and tubes) on the market and making the wrong decision can lead to failure of amplification, lost results, and project delays. This article can help you choose the right PCR plastic consumable to obtain accurate and reliable experimental data from your thermal cycler or qPCR instrument.
When selecting the right PCR plastic consumables, it’s crucial to answer a set of questions that pinpoint your requirements.
The format of the PCR/qPCR plastic consumable that you select often hinges on how many reactions you plan to perform simultaneously. For lower throughput applications, individual PCR tubes are typically preferred. In contrast, for experiments necessitating a broader sample processing capacity, PCR plates are the better option.
PCR tubes are most effective if you conduct only a small number of reactions daily, allowing you to utilize just what you need and easily adjust the number of samples. A PCR tube strip, which consists of 8 or 12 connected tubes, simplifies handling compared to isolated tubes, and they can be cut to fit your sample requirement.
For cases requiring intermediate throughput, 24-well and 48-well PCR plates provide a practical format without necessitating a full plate, maintaining standard reaction volumes while being easier to handle and seal compared to multiple tubes. The Thermo Scientific Thermo-Fast 24-well PCR plate is an excellent choice for those using lower-throughput thermal cyclers or for those with a 96-well cycler wanting to avoid using a full plate for modest sample numbers.
Figure 1. Segmented PCR plates can be separated into 24-well, 32-well, or 48-well sections, or 8-tube strips.
PCR plates serve as the backbone in molecular biology laboratories when handling numerous samples and controls concurrently. These plates come in 96- and 384-well variants; generally, 96-well plates are sufficient for most lab applications, while 384-well plates are advantageous for high-throughput needs by using reduced reagent volumes per well. However, they do necessitate meticulous pipetting and sample management, typically paired with automated liquid handlers.
While most PCR tubes and plates are compatible with both thermal cyclers and qPCR instruments, compatibility alone does not guarantee optimal results. As amplification and analysis occur within the same container in real-time quantitative PCR, selecting a qPCR tube or plate that excels in both functions is essential.
For real-time/quantitative PCR (qPCR), choosing plates with white wells over traditional clear polypropylene is advisable. White wells can improve assay sensitivity, reduce variability in qPCR outcomes, and minimize sample crosstalk while maximizing the amount of fluorescence detected. These features significantly enhance the detection and quantification of fluorescent signals in qPCR reactions.
The use of optical caps and seals plays a crucial role in qPCR. The high-clarity materials optimize fluorescent signal transmission, ensuring maximum fluorescence reaches the detector in your qPCR instrument.
Figure 2. Comparison of qPCR results between clear and white well qPCR plates. (A) White wells provide higher final fluorescence signals with lower Ct values, indicating improved sensitivity compared to clear wells. (B) White wells also demonstrate lower variability than clear wells.
For low-throughput qPCR procedures, optical PCR tubes and caps serve as a smart option, boosting fluorescence signals through frosted sides and optically clear caps, similarly to white qPCR plates.
Once you've determined whether to use PCR tubes, tube strips, or plates, the next critical step is ensuring that your chosen PCR consumables are compatible with your thermal cycler or qPCR instrument. Several characteristics influence compatibility; to identify specific consumables that suit your instrument, consider using the PCR and qPCR plastic consumable selection tool.
Well volume affects the selection of both PCR plates and tubes. Common well volumes include 0.2 mL (standard) and 0.1 mL (low profile). Low profile options generally use less reagent per reaction but may be less efficient for amplifying low DNA levels.
Instrument compatibility heavily relies on the cap style of the PCR tube. Thermal blocks may accept both domed and flat-capped tubes, but opting for a consumable that matches the block design is best. For instance, using flat-capped tubes in a thermal cycler designed for domed caps could lead to inefficient contact between the lid and thermal block.
When selecting PCR plates, the skirt type is vital for compatibility. The skirt refers to the material encircling the edges of a PCR plate; the design of your PCR instrument will dictate whether it accommodates full, semi, or no skirt plates.
The skirt provides stability and influences compatibility. Full skirt plates completely enclose the PCR wells, while the bottoms of semi-skirted plates are visible, and non-skirted plates are more flexible but require support from a PCR rack. More details can be found on Thermo Scientific PCR plates.
For more about skirt styles, low-profile plates, and compatibility, be sure to watch the accompanying video.
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When searching for automation-compatible PCR plates, consider the skirt style previously discussed. Semi- and fully skirted plates are generally compatible with robotic systems; however, fully skirted plates are preferred due to their enhanced mechanical strength and stackability.
The materials used for PCR plates are also critical for automated workflows; seek plates featuring polycarbonate frames for durability combined with polypropylene wells for efficient heat transfer. Thermo Scientific Armadillo PCR plates, with their robust frames and available barcoding, are specifically designed for automated processes.
Spotlight article: Four key features for high-throughput PCR plates
In conclusion, PCR plastic consumables are essential to successful experimental setup and data capture. By dedicating time to evaluate your specific requirements, you can streamline your process to achieve optimal PCR and qPCR results.
Figure 3. Standard- and low-profile PCR/qPCR plastics.
This blog post is the third in a 4-part series on PCR primers. Additional posts are available on understanding PCR primers, designing quality primers, and storing and using primers.
PCR primers are fundamental components in any PCR setup, acting as key players in all PCR-based Learning Labs. The initial step in a successful PCR experiment is to create quality primers. Nevertheless, a common inquiry involves the source of these primers.
In straightforward terms, you order primers from a supplier. Companies like IDT, ThermoFisher, and MilliporeSigma allow you to enter your desired DNA sequence on their websites and will synthesize the custom sequence for you. However, keep in mind that these companies may not sell directly to individuals or ship to home addresses, necessitating further research if you are engaged in home-based biology work.
The primer synthesis is executed through solid-phase chemical synthesis, a method where nucleotides are sequentially added to form the growing chain. While the technical process may seem complicated, machines handle the majority of this work automatically.
When visiting a company’s website for primer ordering, expect to encounter a multitude of options. Here’s a breakdown of critical inquiries you’ll need to address:
What are you ordering? Typically, when ordering primers, numerous options may arise such as labeled primers, DNA probes, and oligonucleotides. For most PCR applications, the best choice is basic DNA oligonucleotides.
The term “oligo” originates from a Greek word meaning “few,” making oligonucleotides essentially “a few nucleotides.” It’s common in scientific circles for “oligonucleotides” or simply “oligos” to be used interchangeably with “primers.” While oligonucleotides frequently serve as PCR primers, they have various applications involving short single-stranded DNA molecules, hence the broader terminology.
What sequence do you want your oligos to have? When ordering primers, you typically input the DNA sequence directly. Be sure both sequences are entered in the 5' to 3' direction, bearing in mind you may view your reverse primer in its reverse complement or 3' to 5' format. For further assistance on designing primer sequences, reference our recent post on how to design PCR primers.
How much do you want to order? Primers are often sold by the nanomole, and unless you plan on conducting numerous PCR experiments, it is advisable to choose the smallest quantity available. Generally, the minimum primer amount (like a 25 nanomole scale from IDT) suffices for approximately 500 reactions depending on your concentration and reaction size.
Due to inherent variability in chemical reactions, the amount of oligos received may fall within a specific range rather than being an exact figure. The exact quantity produced will be reported upon arrival of your primers.
Before finalizing your order, remember primers are surprisingly cost-effective, usually costing under $10 each. In fact, shipping fees can often surpass the prices of the primers themselves, making it advantageous to order in bulk rather than piecemeal.
Contributed by Bruce Bryan, curriculum director at miniPCR bio.
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