Fluorescent quantitative PCR is favored by more and more molecular biology researchers because of its convenient operation, fast operation and accurate experimental results. Today, when the experimental technology is mature, perhaps the most difficult thing for you is not the technical problem of the experiment - but which kind of PCR machine to choose - you need to consult the members of the laboratory, analyze the laboratory now and in the future The needs, balance the budget of the laboratory, and more detailed research on all kinds of tempting promotional materials.
In the face of a variety of different performance products, how do you choose? First of all, I suggest you come out to understand the two misunderstandings of real-time PCR:
Myth 1: The more instrument channels, the better.
With the maturity of PCR technology, multiplex amplification is becoming more and more lively, and the real-time PCR instrument is not immune. From the initial development of ABI's single-channel real-time PCR instrument to the introduction of 4-channel, 5-channel or even 6-channel real-time PCR instruments, the dazzling choices are at a loss, and some people accidentally walked into the channel. The more the better, the misunderstanding.
In order to ensure the accuracy and accuracy of the experimental results, ROX (a fluorescent dye) or a dedicated Reference dye must be used in the experiment when using some manufacturers' 5-channel fluorescence quantitation. These fluorescent dyes must be used separately. aisle. In this way, there are only four effective channels for truly detecting multiplex PCR fluorescence signals, and the use of calibration dyes may increase the cost of later use. Some of the detection channels of real-time PCR are only open for the proprietary fluorescent dyes or reagents of their own manufacturers, and the effective detection channels are not as much as claimed in the publicity materials. It is especially important to confirm the effective detection channel of the real-time PCR instrument before purchase. You can't just listen to the publicity. Considering the number of channels of multiplex PCR, it should also be based on the actual situation in the laboratory. Multiplex PCR is not suitable for everyone, and it is available to everyone because it complicates the experiment.
Myth 2: Real-time PCR machine does not require gradient function
For the quantitative PCR reaction using the dye method, although various PCR primer design software or empirical formulas are used to calculate the melting temperature (Tm value), the formulas used are different, the primer sequences are different, and the difference in Tm values ​​is large. The melting temperature of the primer determines the annealing temperature. Moreover, the combination of bases in the template is ever-changing. For special fragments, the data obtained by the empirical formula may not be able to produce accurate results. The slight difference in annealing temperature may have a decisive influence on the results, so the "conditions" are once Very headache. The appearance of gradient PCR solves some problems. During the reaction, the temperature control conditions of each well can be changed according to the gradient within the specified range. According to the results, the most suitable reaction conditions can be found in one step.
Not only the annealing temperature, but also the denaturing temperature and the extension temperature can be optimized - this is very important for the amplification of many polymerase mixed enzymes such as Invitrogen, Clontech, Promega, most of the high-fidelity Taq enzymes, because Taq and the correct enzyme are optimal. There may be significant differences in reaction temperatures, and optimizing the extension temperature is important.
Chuangsai Technology provides you with high quality fluorescent quantitative PCR:
Fluorescence quantitative PCR with gradient function can complete the optimization process that can be completed in many experiments in the past, which simplifies the cumbersome experiment of exploring the PCR reaction conditions, saves the experiment time and improves the efficiency, and saves the experiment cost.
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