Content1. What is PCR plate?
1.1 What is PCR?
1.1.1 First-generation PCR
1.1.2 Second-generation PCR
1.1.3 Three-generation PCR2. Types of PCR plate
2.1 96 well PCR plate
2.2 384 well PCR plate
2.3 Non-skirted PCR plate
2.4 Semi-skirted PCR plate
2.5 Full-skirted PCR plate3. How to choose PCR plate?
3.1 Why are PCR consumables usually made of PP?
3.2 How to choose between different volumes of PCR tubes/plates?
3.3 Why are some PCR plates covered in flat but some in convex?
3.4 How to choose the colors of PCR plates?4. How to purchase PCR plate?
What is PCR plate?
is a kind of carrier for Polymerase Chain Reaction (PCR)
to carry primers like Taq DNA
polymerase, dNTP, template nucleic acid, Mg, buffer, etc., which are mainly involved in amplification reactions in PCR.PCR plates
are widely used in the fields of genetics, biochemistry, immunology, medicine, etc. They are not only applied to basic research such as gene isolation, cloning, and nucleic acid sequence analysis, but also are used for the diagnosis of diseases or any place where there is DNA or RNA, and are disposable consumable items in laboratories.
What is PCR?
As a molecular biology technique, polymerase chain reaction (PCR) is used to amplify specific DNA fragments. It can be thought of as a special DNA replication outside the body of a living organism. The most important feature of PCR is the ability to amplify minute amounts of DNA dramatically. Thus, no matter in fossilized paleontology, the remains of a historical figure, or the hair, skin, or blood left behind by a murderer many years ago, as long as a tiny bit of DNA can be isolated, PCR can be used to amplify the DNA and compare it. This is where the power of "trace evidence" lies.
It was first conceived by Mullis in 1983 and invented in 1985 as the polymerase chain reaction, which meant the real birth of PCR technology. In 1976, the Chinese scientist Qian Jiayun, who discovered the stable Taq DNA polymerase, made a fundamental contribution to the development of PCR technology. It has been nearly 40 years since the invention of PCR technology, and PCR technology has developed to the third generation. In the past nearly 40 years, PCR technology has made an indelible contribution to the development of life sciences.
After inventing PCR technology, Mullis described it as follows: "With a single molecule of DNA, PCR can produce 100 million similar molecules in an afternoon. All that was needed was a tube, some simple reagents, and a device for heating."
The initial PCR technique used a common PCR amplifier to amplify the target gene, and then the products were analyzed by agarose gel electrophoresis, which can only be done qualitatively.
It is possible to increase trace amounts of DNA
significantly. For the detection of paleontological remains in fossils, hair, skin fragments, or blood from crime scenes, as long as a tiny bit of DNA can be isolated, it can be amplified and compared by using PCR.
Advantages of first-generation PCR
PCR is a molecular biology technique used to amplify a specific DNA fragment, which can be considered a special DNA replication outside the organism.
Disadvantages of first-generation PCR
a. The nucleic acid dyes used in the original PCR technique will cause harm and contamination to the laboratory personnel and the environment.
b. PCR needs to be opened and tested after completion, which is prone to contamination and false-positive results.
c. The assay is time-consuming, troublesome, and error-prone.
d. Only qualitative testing can be done
The second generation of PCR is called fluorescent quantitative PCR technique (Real-Time PCR
), as well as qPCR
. Fluorescence PCR monitors the accumulation of amplification products by adding fluorescent probes to the reaction system that can indicate the reflective process and by the accumulation of fluorescent signals. The results are judged by fluorescence curves and can be quantified with the help of Cq values and standard curves.
For infectious disease pathogen detection, disease drug resistance gene research, drug efficacy assessment, genetic disease diagnosis, and so on.
Advantages of second-generation PCR
a. Low contamination risk.
b. Operation process is simpler, more economical, and more efficient than before.
c. Requires fewer DNA and RNA adding samples.
Disadvantages of second-generation PCR
a. Cq values from different manufacturers' reagents and equipment are not comparable.
b. The effect of background values makes the results easily biased.
c. It is difficult to detect low-copy DNA.
d. Conventional PCR systems are often affected when PCR inhibitors are present in the reaction system.
The third generation of PCR technology is called Digital PCR, dPCR, or Dig-PCR. This is a new PCR assay that detects and quantifies nucleic acids. It uses direct counting of target molecules without relying on any calibrator or appearance, which allows the determination of the absolute number of target molecules to be detected down to a single copy.
For the detection of a small number of mutation-containing cells in a large population of normal cells. By diluting sample DNA into the corresponding assay wells, after PCR amplification, a specific fluorescent probe is added to each well to hybridize with the product, and then the number of mutant and wild-type alleles in the sample is counted directly. The main applications are mutation analysis, allelic deletion, cancer detection of mixed DNA, etc.
Advantages of three-generation PCR
a. Higher sensitivity
Digital PCR splits the traditional PCR reaction into tens of thousands of systems. These systems are independently amplified and independently detected, ensuring that a single-digit number of templates can be detected.
b. More accurate quantification
Digital PCR directly counts the initial number of templates in a reaction by counting the fluorescence of the microsystems. Even if there is more than one template in some microdroplets, it can be calculated by Poisson distribution.
c. Better anti-interference ability
During the distribution of the reaction system in the first step of digital PCR, the template and inhibitor are assigned to different systems to reduce the interference of inhibitors. Unlike qPCR, dPCR detects the endpoint fluorescence, and even if the microsystem is slightly affected, it will not affect the interpretation of the final result.
Disadvantages of three-generation PCR
a. High template quality requirement
Since digital PCR splits reactions into tens of thousands of independent systems, it has relatively high requirements for the amount of template. The amount of template exceeding the amount of microsystem will lead to failure to quantify, and with few amounts will lead to low signal and reduce the accuracy of quantification.
b. Judgment of non-specific products
Digital PCR observes the endpoint fluorescence in each system. Whether the PCR product is specific or not, as long as the fluorescence signal is strong enough, it is also interpreted as a positive result. Therefore, the primer specificity of dPCR is extremely demanding.
Types of PCR plate
PCR plates are mainly made of polypropylene (PP), which makes them better adapted to repeated high and low-temperature settings during PCR reactions, and can be used with autoclave operations. To achieve high throughput operation with row guns and PCR instruments, 96 well PCR plate and 384 well PCR plate are more commonly used. There are different kinds of skirted plates. The skirt provides good stability for pipetting during reaction system construction and good mechanical strength when performing automated mechanical handling. According to the design of the skirt, the PCR plate can be divided into non-skirted PCR plate, semi-skirted PCR plate, full-skirted PCR plate, and fast or low-profile semi-skirted plate.
96 well PCR plate
(96 PCR plate / skirted 96 well plate / applied biosystems 96 well plate)
384 well PCR plate
(384 PCR plate / PCR plate 384 / applied biosystems 384 well PCR plate)
Non-skirted PCR plate
There is no surrounding panel of non-skirted PCR plate. This plate is adaptable to most PCR instruments and real-time PCR instrument modules but is not suitable for automation applications.
(real time pcr plate / rt pcr plate / qpcr 96 well plate / qpcr 384 well plate )
Semi-skirted PCR plate
The semi-skirted plate, also known as half skirted PCR plate, has a short edge around the edge of the plate. This provides adequate support during pipetting and mechanical strength for machine handling.
(96 well pcr / qpcr plate / skirted pcr plate)
Full-skirted PCR plate
The full-skirted PCR plate has an edge panel that covers the height of the plate. This plate format is suitable for PCR instruments with protruding modules and allows for a safe and secure fit. The full skirt also enhances mechanical strength, making it suitable for use in automated workstations.
(full-skirted 96 well plate / taqman array plates / white PCR plates)
How to choose PCR plate?
As we all known that PCR is a basic experimental method in biochemical laboratories. The experimental results, however, are always unsatisfactory, possibly due to contamination of trace amounts of PCR plastic consumables, or experimental interference caused by the introduction of inhibitors. Many reasons affect the results of PCR experiments:
a. Primers -- they are the key to PCR specificity. The specificity of PCR products bases on the degree of complementarity between primers and template DNA.
b. Enzymes and their concentrations.
c. Quality and concentration of dNTP.
d. Nucleic acid of a template (target gene).
e. Concentration of Mg2+.
f. Temperature & time settings.
g. Number of cycles times.
h. Equipment, consumables, etc.
Among them, consumables are a very important and easily overlooked factor.
Why are PCR consumables usually made of PP?
PCR/qPCR consumables are generally made of polypropylene (PP) because it is a biologically inert material. The surface of this material does not adhere easily to biomolecules and has good chemical resistance and temperature tolerance. These materials are often in direct contact with reagents or samples and therefore require high-quality materials and good processing during the production and preparation process.
How to choose between different volumes of PCR tube plate?
Most PCR tubes are available in volumes that meet the requirements of PCR reactions. However, low-volume tubes are preferred for meeting the experimental requirements. This is because low-volume tubes/plates have less space above, which improves the heat transfer rate and reduces evaporation. Moreover, when adding samples, it is necessary to avoid adding too much or too few samples. Overdosing can lead to reduced thermal conductivity, spillage, and cross-contamination. Too little sample may result in loss of sample evaporation.
Common reaction tube sizes and volumes
* Single tube / coupled tube: 0.5mL, 0.2mL, 0.15mL
* PCR plate: 0.1 ml pcr plate / 0.2 ml pcr plate / 24 well pcr plate / 96 well qpcr / 384 well pcr / … …
Why are some PCR plates covered in flat but some in convex?
PCR plate cover in flat provides precise fluorescence signal transmission for q9PCR and is easy to write markers.
Convex cover contacts with the hot cover of the PCR instrument to reduce pressure-induced deformation of the reaction tube. However, it will affect the fluorescence signal transmission and cannot be used in qPCR experiments.
How to choose the colors of PCR plates?
For common PCR reactions, either clear or colored PCR tubes can be used. Colored PCR tubes are more useful for sample sorting and management.
Since qPCR requires real-time quantitative detection of fluorescence signal intensity, sensitive and accurate fluorescence signal transmission is very necessary. The white PCR plates maximize the reflection of fluorescent signals and reduce cross-contamination of signals between wells, which can optimize the results of real-time fluorescent quantitative PCR.
How to purchase PCR plate?
ANTITECK provide lab equipment, lab consumable, manufacturing equipment in life sciences sector.
If you are interested in our PCR plate or have any questions, please write an e-mail to firstname.lastname@example.org, we will reply to you as soon as possible.