Optical quantitative PCR is favored by more and more molecular biology researchers because of its convenient operation, fast running speed and accurate experimental results. Today, when the experimental technology is mature, perhaps the most rare thing for you is not the technical problem of the experiment-but which kind of fluorescent quantitative PCR instrument to choose-you have to consult the members of the laboratory, the analysis laboratory is now and even the future Needs, balance the budget of the laboratory, and study all kinds of enticing publicity materials in detail. Faced with a variety of different performance products, how should you choose?
First of all, I suggest that you come out of two misunderstandings about real-time quantitative PCR:
Myth 1: The more instrument channels, the better
With the mature application of PCR technology, multiple amplification is becoming more and more lively, and the fluorescent quantitative PCR instrument is not immune. From the development of the single-channel fluorescence quantitative PCR instrument originally launched by ABI to the fact that various manufacturers have launched 4-channel, 5-channel or even 6-channel fluorescence quantitative PCR instruments, the dazzling choices make people feel at a loss. More is better "misunderstanding.
When using a 5-channel fluorescence quantifier from some manufacturers, 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. Each of these fluorescent dyes must use a separate detection aisle. In this way, there are only 4 effective channels that can truly detect multiple PCR fluorescent signals, and the use of calibration dyes may increase the cost of later use. Some fluorescent quantitative PCR detection channels are only open for special fluorescent dyes or reagents of their own manufacturers, and effective detection channels are by no means as many as claimed in publicity materials. It is particularly important to confirm the effective detection channel of the fluorescent quantitative PCR instrument before purchase, and you cannot just listen to the publicity.
When considering the number of multiplex quantitative PCR channels, it should also be based on the actual situation in the laboratory. Multiplex PCR is not suitable for everyone and is available for everyone, because it complicates the experiment.
Myth 2: Real-time PCR does not require gradient function
For quantitative PCR reactions using the dye method, although there are various PCR primer design software or empirical formulas to calculate the melting temperature (Tm value), the difference in the Tm value will be very different depending on the formula used and the primer sequence. 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 produce accurate results. The slight difference in annealing temperature may have a decisive effect on the results. Therefore, "touching the conditions" was once Very headache. The emergence of gradient PCR partially solved 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 denaturation temperature and extension temperature can be optimized-this is very important for many high-fidelity Taq enzymes such as Invitrogen, Clontech, Promega, etc., which are the best for Taq and correction enzymes. There may be significant differences in reaction temperature, and it is important to optimize the extension temperature.
The use of gradient quantitative fluorescent quantitative PCR can complete the optimization process that can be completed by multiple experiments in the past, simplifying the tedious experiment of exploring the PCR reaction conditions, not only saving experimental time and improving efficiency, but also saving experimental costs.
When we get out of the misunderstanding and want to choose a fluorescent quantitative PCR instrument that best suits our needs, what should we pay attention to?
1. The detection flux of the instrument
When purchasing a quantitative PCR instrument, choose different throughput instruments according to the actual needs of the experiment. At present, the detection throughput of fluorescent quantitative PCR on the market is as small as 16 to 384. If you conduct general gene expression research or pathogen detection, you really do nâ€™t need to buy expensive instruments, and the 96-well flux is sufficient; laboratories that need to find new drug targets and disease markers can consider buying instruments with 384-well plates. If a laboratory with limited funds cannot purchase a 384-well plate instrument, you can consider purchasing a 96-well plate quantitative PCR machine that runs fast (with a FAST module). With high-throughput instruments, you will find sample preparation and the construction of small systems and multi-sample PCR systems as flasks that limit experiment speed and results. Roche's MagnaPure accounting purification system can be used with Roche's Lightcycyler fluorescent quantitative PCR instrument. Eppendorf's ep Motion5070 and 5075 fully automatic workstations can solve the problem of nucleic acid purification, and can also construct PCR reaction systems of 96-well plates and 384-well plates, without adding samples to the eyes and hand cramps.
2. Features of hardware design
Fluorescence quantitative PCR instruments mainly include traditional 96-well plate type, innovative centrifugal type, etc. Each design has its own uniqueness but also has unavoidable shortcomings.
Traditional 96-well fluorescence quantitative PCR can accommodate a large sample size, and no special consumables are required. Some traditional 96-well plate instruments use halogen tungsten lamp excitation and CCD detection. These optical structures are on the top of the 96-well plate. Each sample hole has a different optical path from the light source and the detector-the edge effect produces the results. influences. In order to ensure accurate and accurate experimental results, such instruments usually use ROX (a fluorescent dye) or a special Reference dye as the reference to correct the experimental results during the experimental process. The short service life and high replacement cost of halogen lamps have become a headache for many users. During the experiment, the tungsten halogen lamp is used as a thermal light source, and generating more heat energy has a certain influence on the experimental results. The biggest advantage of CCD is that it can scan the fluorescence signal in all samples at the same time, but the sensitivity is low, and there is interference between the fluorescence signals detected at the same time. Fluorescent quantitative PCR produced by companies such as ABI and Bio-rad belongs to this category. However, Bio-rad's IQ series of fluorescent quantitative PCR has a gradient function.
Innovative centrifugal instruments usually use LED excitation and PMT detection. The centrifugal design avoids edge effects. The LED light source is a cold light source that has no effect on the experiment, so there is no need to use other fluorescent dye calibration instruments, and the long service life does not require frequent replacement. PMT can only collect a single fluorescent signal at a time, but the detection sensitivity is high. However, this type of instrument runs very fast, but it also has the disadvantages of small sample size, expensive consumables, no gradient function, and time integration. Corbett's Rotor-gene series and Roche's Lightcycler series are all such instruments.
With the use of fluorescent quantitative PCR technology, smart instrument manufacturers have made great improvements on traditional 96-well plate instruments. The first is that Stratagene's Mx3000p breaks through the use of CCD and uses PMT to detect fluorescent signals, which greatly improves the sensitivity of the experiment, but the excitation light source still uses the traditional tungsten halogen lamp and has no gradient function. The Mastercycler ep realplex launched by Eppendorf is a fluorescent quantitative PCR instrument with gradient function. It uses 96 LEDs as the excitation light source. It uses advanced CPM (second-generation PMT) and 96-in-1 optical fiber as the detection system, which not only avoids The edge effect also ensures that there is no interference in the fluorescence signal between all samples.
3. Running speed
Among the many technical parameters of the fluorescent quantitative PCR instrument, the temperature rise and fall speed is also an indicator that manufacturers like to vigorously promote. Faster temperature rise and fall can shorten the reaction time, and shorten the possible non-specific binding and reaction time, and improve the specificity of PCR. In order to better complete the experiment, various manufacturers have launched fluorescent quantitative PCR instruments that can run quickly. For example, Lightcycler 2.0 introduced by ROCHE uses aerodynamic principles to complete 30-40 cycles in about half an hour. ABI's 7500 can be equipped with a FAST module with a temperature rise and fall speed of 5 Â° C / sec. It can complete 30-40 cycles in about 40 minutes. Recently, eppendorf's newly launched Mastercycler ep realplex 4S and 2S silver module fluorescent quantitative PCR instrument, the temperature rising and falling speed can reach 6 â„ƒ / second and 4.5 / second, which is the fastest fluorescent quantitative PCR instrument in the same kind of products at present. A PCR reaction that originally required 40-60 minutes on other instruments, eppendorf Mastercycler ep realplex 4S and 2S (silver modules) only need to run for 28 minutes-don't underestimate the tens of minutes saved, you can run more in one day Several rounds.
Large-scale busy laboratory equipment is certainly enviable, but conventional laboratories can also have wonderful choices. The current instrument suppliers have many technical experts, and some can also provide a basic research platform for the entire molecular biology, not only to understand and meet your current needs, but also take into account your changing needs-upgrades and replacement modules. Bio-rad's Mycycler can choose to upgrade the module to a quantitative PCR instrument. ABI's 7900 can choose to change the 96-well slot to a 384-well slot. Rotor-gene3000A, a centrifugal two-channel Rotor-gene3000A, can be upgraded to a four-channel quantitative PCR system according to your research needs. Eppendorf is even more considerate in this regard. For example, the laboratory already has a Mastercycler ep that can be upgraded to a Mastercycler ep realplex fluorescent quantitative PCR instrument according to your requirements, and there are 4 models that can be upgraded: 2 channel silver, aluminum module And 4-channel silver and aluminum modules. You can purchase or upgrade any Mastercycler ep realplex fluorescent quantitative PCR instrument according to your own funds and experimental needs.
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