The same is done for PCR, why is the result always so much worse than the brother?
In the laboratory, I looked at the PCR performed by my brother. The amplification efficiency was high and the electrophoresis bands were perfect. Then I looked at my own. After working hard for a long time, I felt that the reaction was quite good. The result was a false positive test... What is the problem? ?
The primer design is no problem, and the operation is very careful. It is not a pot of template quality, and the temperature is well controlled...
I believe that many partners have also encountered various problems. When we hit a wall in a PCR experiment, we subconsciously analyze the factors in the reaction system, but we often ignore the important influence of consumables in the experiment.
Your PCR result is wrong, PCR consumables may be the essential reason. On the one hand, trace contamination of consumables or the introduction of inhibitors will cause experimental pollution; on the other hand, improper selection of consumables will also make the experimental results suffer.
The problem is coming again. There are many types of PCR consumables, such as a series of PCR and qPCR plates such as 8-strip tubes, low-volume tubes, no skirts, half skirts, rising holes, etc. How to choose the right one to make the experiment smoother? What are the issues that need attention...Thinking about it makes people bald~
Don`t worry, today I have compiled the common problems of PCR consumable selection based on many years of experience. I hope it can help everyone. Friends are also welcome to leave a message to share their experience or ask questions~
Q1: Why are PCR consumables generally made of PP?
Yongyue: Because PCR/qPCR consumables are usually in direct contact with reagents or samples, and polypropylene (PP) is a biologically inert material, the surface is not easy to adhere to biomolecules, and it has good chemical resistance and temperature resistance ( It can be autoclaved at 121 ℃, and can also withstand temperature changes during thermal cycling).
Q2: Different volumes of PCR tubes/plates, how should I choose?
Yongyue: The purpose of selection: select appropriate products according to specific experimental requirements. Most of the volume of the PCR tube can meet the requirements of the PCR reaction. On this basis, it is recommended to prefer low-capacity tubes. Because the low-volume reaction tube/plate has a small upper space, it can increase the thermal conductivity and reduce evaporation. At the same time, it is necessary to avoid adding too much or too little when adding samples. Too much will result in a decrease in thermal conductivity, overflow and cross-contamination, while too little may cause sample evaporation loss.
The specifications and volume of common reaction tubes:
Single tube/tube strip: 0.5 mL, 0.2 mL, 0.15 mL
96-well plate: 0.2 mL, 0.15 mL
384-well plate: 0.04 mL
Q3: When the sample volume is small, I will choose single tube or header tube, but why are some flat caps and some convex caps?
Yongyue: When the sample volume is small, single tube or combined tube will be preferred. However, when the reaction volume is large, a single tube is dominant, and the volume can reach 0.5 mL. The flat cover and convex cover have their own advantages, please refer to the following information for details.
Single tube: 0.2ml and 0.5ml, the number of samples can be selected flexibly.
Connecting tube: 0.2ml or 0.15ml optional, 8 or 12 connecting tubes are common.
Flat cover: It can provide accurate fluorescence signal transmission for qPCR, which is convenient for writing and marking.
Convex cover: Contact with the thermal cover of the PCR machine to reduce the deformation of the reaction tube caused by pressure, but it will affect the fluorescence signal transmission and cannot be used in qPCR experiments.
Q4: How do I choose PCR tubes/plates of different colors?
Yongyue: It is possible to use transparent or colored PCR tubes for ordinary PCR reactions, and colored and transparent PCR tubes are more helpful for sample classification management.
However, qPCR recommends the use of high-quality white PCR consumables. Since qPCR requires real-time quantitative detection of fluorescence signal intensity, it needs sensitive and accurate fluorescence signal transmission. Experiments have proved that, compared with traditional transparent PCR products, white PCR products can reflect fluorescent signals to the greatest extent, reduce cross-contamination of signals between wells, and can optimize the results of real-time fluorescent quantitative PCR.
Q5: The thickness of the PCR plates/tubes I bought before is different. Is the thicker one more stable?
Yongyue: No, the thickness of the tube wall directly affects the thermal conductivity. The extremely thin wall thickness can optimize heat transfer and reduce cycle time. If you want to improve the efficiency of the PCR reaction and ensure that the reaction proceeds stably and efficiently, PCR consumables with uniform and ultra-thin walls are the best choice.
Q6: For 96-well or 384-well plates, why some have skirts and some do not?
Yongyue: Actually, the skirt of the PCR/qPCR plate is to better adapt to automation applications, which can provide stable support and mechanical resistance to the instrument, and the stability during pipetting will be higher.
PCR panels are generally divided into no skirts, half skirts, and full skirts.
No skirting board: It can be used for most PCR machines or qPCR machines, but it is not suitable for automation applications. The stability is not high during the pipetting process, and it needs to be used with the plate holder.
Semi-skirt board: can be adapted to labels or barcodes, and automated applications, and has good pipetting stability.
Full skirt board: very suitable for automated experimental applications, and can also be adapted to labels and application barcodes. The mechanical strength is good, and it can be applied to the thermal cycler of the protruding module, and the stability is high during the pipetting process.
Q7: For 96-well or 384-well plates, why are the cut corners and labels not the same?
Yongyue: This is determined by the role of corner cutting and marking.
Cut corner: The choice of the cut corner position of the PCR plate depends on the requirements of the adapted instrument, which is convenient for positioning.
Labeling: The alphanumeric labeling of the PCR plate helps to identify individual wells and sample locations. Generally, it is a protruding color digital logo or an imprinted logo. For some automated application experiments, the sealing performance of the reaction plate with imprinted marks will be better.
Q8: Why are some PCR plate holes protruding, while others are flat? Which is better?
Yongyue: The right one is the best. Common PCR plates have flat hole edges and raised hole edges.
The reaction plate with a flat edge is suitable for most thermal cyclers.
The reaction plate with the rising edge of the hole facilitates sealing and reduces the risk of cross-contamination between samples.
Q9: There are many types of PCR sealing films. Which experiment is more effective?
Yongyue: According to the specific PCR experiment requirements, the suitable high-quality sealing film is selected, which can not only effectively protect the samples and prevent contamination, but also provide reliable experimental test data.
Yongyue provides high-quality PCR/qPCR membranes with strong temperature tolerance and sealing, high transparency, and easy operation.
Yongyue Medical has many years of production and processing experience and high industry standards for PCR products. The product line is rich in variety, which can meet most of the PCR experimental applications. Come and choose the laboratory consumables suitable for your experimental applications.
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