Today, let's talk about a key element in PCR experiments - consumables. As a frequent visitor to PCR, it is necessary to understand the characteristics and terminology related to PCR consumables.
There are a wide variety of PCR consumables on the market, including brands, specifications, colors, etc., which are dazzling. Let's understand it step by step.
pcr plate
1. Materials
Usually, the raw material of PCR consumables is polypropylene. It is a good heat-resistant inert material. The working temperature range is -30 to 140 °C, which enables it to withstand the thermal energy changes during the thermal cycle of the PCR reaction, and plays a good role in sealing and protecting the substances in the PCR reaction system.
Learn more about polypropylene
2. Color
Common ones are white tubes and transparent PCR tubes. In traditional PCR experiments, there is no difference between the two. However, in the same real-time/qPCR experiment, white tubes were relatively more sensitive and consistent for qPCR instruments with apical signal reads (see Figures A and B below). why is it like this?
pcr tube
A: lower Ct values for white tubes compared to clear tubes; B: better repeatability with white tubes.
The core data of qPCR is the Ct value, and the fluorescence signal is the data source for calculating the Ct value, so it is very important whether the fluorescence in the tube can be completely transmitted to the detector. The white tube wall prevents the fluorescence from passing through the tube wall and refracting (Figure A below), thereby preventing the fluorescence from being transmitted to the heating block (i.e., the wall of the metal hole where the tube is placed) and being absorbed or inconsistently reflected by the block. A stronger and more concentrated fluorescent signal was collected at the tip of the white tube compared to the clear tube (Figure B below).
pcr tube
A: The changing trend of the fluorescence signal in the transparent tube and the white tube; B: The fluorescence intensity comparison after adding the same amount of fluorescein.
So if you're using a top-reading qPCR instrument, try white tubes.
3. Quality (Lean or Not Lean)
The importance of consumable quality is emphasized here. qPCR consumables must be free of contaminants and inhibitors. High pressure and radiation remove bacteria and DNases, but not dust and DNA residues, which may inhibit PCR reactions or produce nonspecific amplification. Substances have absorbance, and even tiny pollutants will absorb the fluorescence in the reaction system and affect the collection of fluorescence signals. An exaggerated version of the experiment was used to demonstrate the effect of impurity contamination on the experiment. As shown in the figure below, an impurity was placed in one of the reaction wells of the octave. As expected, the amplification curves showed marked anomalies.
Impurities in PCR reaction tubes
↑ Impurities in the reaction tube
8 strips
↑ Repeated reaction results for 8 tubes, the left well impurity "deviates" the curve.
Reminder: Be careful not to expose the consumables to the air for a long time. After taking out the consumables, be sure to seal the packaging bag quickly to avoid touching it with bare hands. If possible, work on a clean bench.
4. Capacity / Size / Flux
The volume determines how much sample (reaction volume) can be loaded. Overdosing may result in reduced heat transfer efficiency, spillage, or cross-contamination; underdosing may result in evaporative sample loss. The heating block of the qPCR instrument will also determine the applicable consumables and the applicable reaction system size. Throughput is easy to understand, it is the maximum number of samples that can be loaded in a single experiment. Common capacity and flux specifications are shown in the table below:
Single tube/eight rows 0.2mL, 0.1mL
96-well plate 0.2mL, 0.1mL
384-well plate 0.2mL
The capacity specifications are also generally divided into standard and low types, as shown below. Low profile consumables have a lower height. This short design minimizes the space above the reaction system ("dead volume" - the useless part), reduces the effects of evaporation during thermal cycling, and enables the heating module to wrap around it to a greater extent to Improve heat transfer efficiency. Therefore, low profile tubes are also known as "fast" tubes or plates.
pcr tube
Reminder: The commonly used reaction volume is 20 µL, and it is recommended to use a small volume reaction tube of 0.1 mL or a small volume.
5. Wall thickness
The thickness of the tube wall directly affects the thermal conductivity. PCR consumables should have uniform wall thickness to provide consistent heat transfer. The ultra-thin tube wall is 50% thinner than the standard version, which further reduces the thermal barrier and brings faster and better response effects. Therefore, in order to maximize the efficiency of PCR reactions and ensure stable and efficient reactions, uniform and ultra-thin PCR consumables are the best choice.
6. Transparency
Caps and sealing films for qPCR require high optical clarity to ensure optimal penetration and transmission of fluorescent signals while reducing distortion (for top-read qPCR instruments). The higher the transparency of the tube wall, it may affect the experimental results. The reason has been explained above. Traditional PCR can ignore this feature.
7. Skirts
The skirt of a PCR plate refers to the peripheral extension of the plate. When building reaction systems, the skirt acts as a stable support for pipetting processes and provides mechanical strength and grip when combined with automated workstations. There are three common types of skirts, half skirts and full skirts (corresponding to A, B, and C in the figure below).
Skirtless: Can be used with most PCR or qPCR instruments, but not for automated applications. Easy to move during pipetting, not very stable, needs to be used with a plate holder.
Half Skirts: Ideal for automated applications with good pipetting stability.
Full Skirt Plate: Ideal for automated applications. Good mechanical strength, suitable for PCR instruments with protruding modules, high stability during pipetting.
pcr plate
Tips: The significance of the skirt is more to facilitate industrialization and automated batch processing, combined with the robotic arm. It may not be needed in traditional research labs, no skirt is sufficient.
pcr plate
8. Parafilm
The PCR plate sealing film is made of clear plastic or aluminum foil. Adhesive transparent films are more common, suitable for 96 or 384 well plates, and should have good enough light transmittance. When sealing the film, a small card will be used to press the film around the sealing hole to avoid air leakage.
Adhesive aluminum films may be less common. Although not suitable for qPCR, it is the ideal consumable for light-sensitive samples. Because it can be punched, it facilitates sample transfer in PCR (for conventional PCR).
PCR plate sealing film
Summarize
As the carrier of PCR system, consumables are very important to improve the quality of experimental data. I hope this sharing can help you understand some tips on PCR consumables and provide help for better selection of consumables.
