#pcr machine

Advanced Gradient PCR Machine

Standard gradient PCR machineis

In the realm of modern scientific research and diagnostics, the PCR machine stands as an indispensable tool that has revolutionized the field of molecular biology. At TekMatic, we take immense pride in providing cutting-edge PCR systems that empower researchers, scientists, and medical professionals to unlock new frontiers in their respective domains. With our unwavering commitment to innovation and excellence, we have emerged as a leading global provider of high-tech engineered solutions, catering to the diverse needs of our esteemed clientele.

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When purchasing a fluorescent quantitative PCR instrument, several key parameters should be paid attention to:

1. The detection throughput of the instrument (Throughput)

The first thing to consider is the frequency with which the laboratory currently needs to use a quantitative PCR instrument. David Ginzinger, head of genome analysis equipment at the University of California, San Francisco Cancer Center, has tested most of the quantitative PCR machines on the market. He said that if it is not used in large-scale batches, for example, it is mainly used to test known genes, then expensive high-end products are really not needed. But if it is those laboratories that need to find new drug targets and disease markers, they need instruments that can accommodate 384-well plates and run faster. However, just such a high-throughput instrument is not fast enough, and you will find that sample preparation becomes a bottleneck for the speed limit. Therefore, high-throughput laboratories often need further automated instruments. As long as the "click", the instrument will automatically process samples, purify nucleic acids, and prepare PCR templates.

2. The ability of multiple amplification

Multiple amplification is getting hotter and hotter. Real-time quantitative PCR machines are not immune. The high-end quantitative PCR instrument has multiple detection channels, so the instrument can detect the amplification process of multiple templates in the same sample tank. The new LightCycler2.0 has 6 detection channels, 3 more than the original. SmartCycler II has 4 detection channels. Stratagene, famous for its library

products, also has a quantitative PCR machine Mx3000P, which is less than half the price of Roche and has a good reputation, which is 4 detection channels. However, multiple amplification is not suitable for everyone, because it complicates the experiment.

3. software

When you are going to choose a quantitative PCR machine, you must check the accompanying software. Is it easy to use? Is it possible to perform the analysis you need? There are several output formats. However, you need to pay attention to whether the software that is too easy to use has simple functions and cannot perform complex calculations. The previous software still requires you to do further analysis. The new software can already handle everything. In addition to basic functions, some software can also have more functions, such as primer and probe design, multiple analysis design, background correction, data normalization, and so on. Willian Demyan, head of the Application and Technical Services Department of Roche's Applied Science Department, emphasized: Quantitative PCR is a geometric amplification process. At the beginning, the difference between one copy and the result is geometrically different.

4. Flexibility

Although the large-scale busy laboratory equipment is advanced, it is enviable, and the conventional laboratory can also have wonderful choices. The current suppliers have many technical experts, not only understand your current needs, but also consider possible changes in your future requirements very thoughtfully, such as providing a variety of upgradeable accessories and modules. BioRad's MyCycler can select modules and upgrade to real-time quantitative. ABI's quantitative PCR instrument has a variety of different sample tanks, which can change a 96-well tank into a double 384-well tank to meet part of the high-throughput requirements. For laboratories that are just starting to do a small amount of qPCR, the SmartCycler II quantitative PCR instrument is a good choice. A basic module has 16 independent temperature-controlled sample tanks, and 16 completely independent different experiments can be performed at the same time or before and after. Access"; when the laboratory needs a larger-scale experiment, it can be upgraded economically, from 1 to 6 basic modules, that is, 96 sample wells. Although a 96-well plate cannot be used, when a student starts a small experiment, others can plug in and start their own independent experiments at any time without interfering with each other, which increases flexibility. It is a very flexible choice for laboratories that only do a few experiments at the beginning and have a tight budget.

5. Uncertainty

 Gradient PCR gene amplification instrument is an instrument and equipment that uses PCR (polymerase chain reaction) technology to amplify specific DNA. It is widely used in medical and biological laboratories, for example, to determine whether a sample will show certain Atlas of genetic diseases, diagnosis of infectious diseases, gene duplication, and paternity testing, etc.

PCR (polymerase chain reaction) is a method of enzymatically synthesizing specific DNA fragments in vitro. It consists of several steps of high temperature denaturation, low temperature annealing, and temperature extension. It has the characteristics of strong specificity, high sensitivity, simple operation and time-saving.

The main function of PCR gene amplification instrument is similar to the basic measuring instrument, and it has higher requirements for measurement elements. Once out of control, the instrument will not work normally, so the PCR gene amplification instrument also needs regular maintenance.

Problems that need attention in the maintenance of gradient PCR gene amplification instrument:

1. The PCR gene amplification instrument needs to be tested regularly, usually at least once every six months.

2. The required temperature of the PCR reaction is inconsistent with the actual distribution of the reaction temperature. When the average temperature difference of each well is found to deviate from the set temperature by more than 1 to 2°C, the temperature correction method can be used to correct the actual PCR reaction temperature difference.

3. The key to the PCR reaction process is the time control of the rising and cooling process. The shorter the requirement, the better. When the cooling process of the PCR machine exceeds 60s, the refrigeration system of the instrument should be checked, and the reaction base should be cleaned for the air-cooled PCR machine. For other refrigeration systems, check the relevant refrigeration components.

A. DNA template:

Try to use high-quality, purified DNA as a template

· When the fidelity needs to be improved, a higher DNA template concentration can be used and the number of cycles can be reduced

· Template dosage: Take 50 μl reaction system as an example——

Human genomic DNA: 0.1~1.0 μg

E. coli genomic DNA: 10~100 ng

Lambda DNA: 0.5~5 ng

Plasmid or viral DNA: 0.1~10 ng

B. Primer design principles:

· The length of the primer must meet specificity requirements, generally between 18 and 25 bases; when amplifying long fragments, it is best to be between 24 and 30 bases;

· When introducing a cloning site, the end of the primer should add more than 3 bases;

· The (G+C)% content should be controlled within 40-60% as much as possible, and the (G+C)% content of the two primers should be as close as possible;

· GC bases are evenly distributed in primers;

· Try to avoid the same base appearing more than three times in a row, and avoid using A or T at the 3'end;

· Avoid primer internal pairing to form a secondary structure;

· Avoid pairing bases between the forward and reverse primers, especially the three bases at the 3'end, otherwise it is easy to generate primer dimers;

· The melting temperature (Tm) of the two primers should be between 42~65℃, and the difference between the two primers should not exceed 5℃;

· Calculation method of primer Tm value:

Below 20 nt: Tm = 2℃ x (A + T) + 4℃ x (G + C)

20 nt or more: Tm = 81.5 + 0.41 x (GC%) -600/nt (nt: base number of primer)

· Primer usage:

· 0.1~1.0 μM, usually starting at 0.2 μM, adjust the dosage according to the system;

· When using degenerate primers or random primers, the total amount of primers needs to be increased to compensate for the loss of yield; however, as the amount of primers increases, the specificity will decrease;

· When the template is larger or larger, or the structure is more complicated (such as human genomic DNA), you need to reduce the amount of primers to improve specificity;

· When the template is small (such as a plasmid template), increasing the amount of primers can increase the yield.

C. Nucleotides (dNTPs):

· The concentration of conventional dNTPs is 0.1~1.0 mM for each nucleotide, usually starting at 0.2 mM, and the dosage can be adjusted according to the system;

· Low concentration (0.05~0.1 mM) can increase fidelity, but will reduce yield;

· High concentration increases yield, especially long-segment PCR, but reduces fidelity.

D. Magnesium ion concentration

· For Taq DNA polymerase, the optimal concentration of magnesium ions is 1.5~2.0 mM;

· The optimal concentration depends on the template, buffer, DNA and dNTPs (each of which may chelate magnesium ions);

· Too low magnesium ion concentration will reduce output;

· Too high magnesium ion concentration will increase non-specific PCR products;

· When optimizing the magnesium ion concentration, it is usually increased by 0.5 mM gradient, up to 4 mM.

E. Taq DNA polymerase concentration

· The recommended concentration is 1~2.5 U/50 μl reaction system.

F. Initial reaction

· Prepare the reaction system on ice;

· Add polymerase at the end;

· After preheating the thermal cycler to the denaturation temperature (94°C), put it in the PCR tube and proceed immediately.

G. Denaturation temperature and time

· The initial denaturation is usually at 94°C, so that the DNA double strands are completely opened;

· Denaturation time is usually 15~30 seconds;

· Avoid prolonged or high temperature incubation;

· Templates with high GC content can increase the denaturation temperature to 98°C.

H. Annealing temperature and time

· Normally, the annealing temperature is the primer Tm minus 5°C, which is between 55 and 60°C;

· Increasing the annealing temperature is conducive to reducing non-specific bands;

· The conventional annealing time is 15-30 seconds.

I. Extended temperature and time

· The extension reaction is usually carried out at 72°C.

· The extension time of Taq enzyme is about 15~30 seconds/kb DNA;

· When the product is less than 1 kb, the recommended extension time is 30 to 60 seconds;

1. The unevenness of refrigeration semiconductor chips

Most of the current PCR machines use semiconductor refrigeration chips to control the temperature. When it is energized, one side is hot and the other side is cold, and the opposite electrode is opposite to the hot and cold side.

Although the semiconductor refrigeration film is industrialized production, almost all of them are produced manually, so each film is different, and large manufacturers will match them according to the needs of users. For example, if a PCR machine requires three pieces, then three pieces will have roughly the same resistance value. The resistance value is generally written by the manufacturer on each refrigeration chip (the measurement is not accurate when using a multimeter by yourself, because the temperature changes when the power is turned on, and the resistance value will change), so the quality of the semiconductor chip will affect the uniformity of the PCR Machine.

2. Number of temperature control points

On the one hand, there is a certain difference in the semiconductor chip itself, on the other hand, the performance will decrease after a period of use, but the degree of decline is not consistent, and the heat dissipation of the opposite side of the edge and the middle semiconductor chip is inconsistent (to be discussed later). Cause the uneven temperature of the module. The commonly used solution is to place a temperature sensor under each semiconductor chip, and have a corresponding control circuit to independently control and calibrate (this will increase the cost of the manufacturer). For example, AB's 9700 has three sensors and corresponding circuits, while the low-end has only one.

Another advantage of multiple sensors is that different temperatures can be controlled separately to generate temperature gradients.

3. Edge effects and mitigation methods

The edge of the module (referring to the metal block where the PCR tube is placed) dissipates heat quickly, and the temperature is generally low, which is the so-called edge effect. In fact, the radiator also has the problem of low edge temperature. To reduce the edge effect, the edge-assisted heating technology is generally used, that is, a circle of resistance wire or heating film for auxiliary heating is added to the periphery of the module. But in fact, the edge effect is not uniform. One is that the temperature at the corners is lower, and the other is also related to the blowing direction of the radiator fan (front and back blowing is uneven front and back, while blowing from bottom to top is uneven center and edge).

4, the heat dissipation uniformity of the radiator

The heat dissipation uniformity of the radiator has a great influence on the temperature uniformity of the module. The semiconductor chip controls the temperature difference between its front and back sides, and each semiconductor chip is controlled as a whole. If the temperature of the heat sink on the back is uneven, of course the temperature of the module on the front will be uneven. The radiator material is theoretically better to use copper, but due to price reasons, it is mostly aluminum alloy with high thermal conductivity. Another aspect is the cooling of the fan. The wind of the fan is large, and the uniformity of heat dissipation will increase, but the noise will also increase greatly. For example, the noise of the AB instrument is very high. How to improve the heat dissipation uniformity of the radiator is also a science.

5, the material and shape of the module

From a uniformity point of view, the module is best made of silver, followed by copper, and then aluminum alloy. The modules generally used in the market are aluminum or silver. The main reason is that the volumetric specific heat capacity of copper is too large, which is not conducive to the rate of temperature rise and fall. Silver has the same problem, so the silver modules on the market are actually hollow. But when it is made hollow, it has a negative effect on temperature uniformity. Therefore, the temperature uniformity of silver module instruments on the market is not higher than that of aluminum alloy, but the temperature rise and fall rate is higher. Because silver is easy to oxidize, some silver modules are gold-plated (don't think it is pure gold or a whole piece of gold or silver), and some are gray after surface oxidation treatment.

The aluminum alloy module also has shape problems. The large volume and the more connections between the holes are good for uniformity, but not good for the temperature rise and fall rate. Various manufacturers have also worked hard on the shape of the module, which is a balance.

6, temperature rise and fall speed

The temperature rise and fall speed is another important indicator of the PCR instrument. Too high a temperature rise and fall speed will also affect the uniformity of the temperature. Because the temperature rise and fall speed is high, the module does not quickly transfer heat, and the temperature difference caused by the difference of the semiconductor chips and the heat transfer will be greater.

In addition, what users want is the temperature in the tube when using the PCR instrument, and what the instrument can measure is the temperature of the sensor under the module. The temperature in the tube calculated by the results of theory and experiment is different from the actual temperature. The faster the temperature rise and fall. , The greater the difference.