Instrument Plus | Instrument Introduction: Nucleic Acid Extraction and Purification _ Operation


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Original Title: Instrument Plus | Instrument Introduction: Nucleic Acid Extraction and Purification Introduction of Nucleic Acid Extraction and Purification Nucleic acid is one of the most basic substances of life, which can be divided into DNA and RNA, and widely exists in all living organisms such as animals, plant cells, microorganisms and so on. It not only plays a role in storing and transmitting genetic information, but also plays an important role in protein biosynthesis, thus playing a decisive role in a series of important life phenomena such as growth, heredity and variation. Nucleic acid extraction includes DNA extraction, RNA extraction and plasmid extraction. Nucleic acid is the carrier of genetic information, the material basis of gene expression, and the main object of molecular biology research. To study the structure and function of nucleic acid, it is necessary to extract and purify the nucleic acid. The nucleic acid extractor is an instrument that uses the supporting nucleic acid extraction reagents to automatically complete the extraction of sample nucleic acid. The nucleic acid extractor is divided into two types: one is a large automatic, generally called automatic liquid workstation; the other is a small automatic nucleic acid extractor, which uses the packaged supporting reagents to automatically complete the extraction and purification process. Large automatic liquid workstations are rarely used because of the high cost of equipment and operation, and they are suitable for extracting thousands of the same kind of specimens at a time; while small automatic instruments are more and more used because of the low cost of equipment and operation and the convenience of operation. Method for extracting and purifying nucleic acid Since nucleic acid was first discovered in 1869, many researchers have made unremitting efforts to explore the extraction methods of nucleic acid, improved various materials and reagents of nucleic acid, and various reagents such as sodium dodecyl sulfonate, phenol, urea and guanidine salt have been applied to nucleic acid extraction experiments, and various commercial kits for nucleic acid extraction have emerged as the times require. The traditional extraction methods include phenol extraction, alkaline lysis, CTAB extraction and EtBr-CsCl gradient centrifugation. These traditional extraction methods can separate DNA and RNA from different tissue samples, but these techniques include precipitation and centrifugation steps, which require a large amount of biological samples, and the extraction steps are more complex, time-consuming and laborious, the yield is not high, it is difficult to achieve automatic operation, in addition, most of the traditional methods also need to use toxic chemical reagents. Therefore, with the development of molecular biology and polymer materials science, the traditional technology of separating nucleic acid from liquid phase system is gradually replaced by the new method based on solid phase carrier. Solid phase carrier adsorption method: The new nucleic acid extraction methods based on solid phase adsorbate carrier mainly include: centrifugal column extraction method, glass bead adsorption method, silica matrix method, anion exchange method and nano-magnetic bead extraction method. The operation steps of this kind of method can be divided into three parts: (1) promoting cell rupture by using a lysis solution to release nucleic acid in a liquid phase; Expand the full text (2) the released nucleic acid is specifically combined on a specific carrier by utilizing the stronger affinity and adsorption force of the carrier to the nucleic acid, so that other impurities such as protein, polysaccharide, lipid and the like are still dissociated in a liquid phase and are removed along with the removal of supernatant, And (3) eluting the nucleic acid adsorbed on the carrier by adjusting the ionic strength and pH value of the eluent to obtain the purified nucleic acid. Among them, the DNA extraction kit by centrifugal column method is widely used in the market because of its low price and relatively convenient operation. However, with the increasing demand for DNA extraction, the shortcomings of DNA extraction by centrifugal column method have become increasingly prominent. The centrifugal column method has the unavoidable disadvantages of large sample demand, large loss and inability to deal with rare samples. At the same time, the centrifugal column method needs repeated centrifugation in the process of DNA extraction, which is not convenient for high-throughput and automatic operation, and is incompatible with the requirements of modern biological experiments. In order to meet the development needs of high-throughput, high-sensitivity and automated operation of modern molecular biology, the DNA extraction technology by magnetic beads was born in the 1990s. Its principle is that the surface of magnetic beads has specific active groups, which can bind specifically and reversibly with nucleic acid under specific conditions. At the same time, the magnetic response ability of magnetic beads is used. Can conveniently carry out directional movement and enrichment under the action of an external magnetic field so as to realize the separation and purification of nucleic acid. Advantages of nucleic acid extraction by magnetic beads DNA extraction by magnetic beads is a perfect combination of nanotechnology and biotechnology, which has incomparable advantages over other DNA extraction methods: 1. Sample demand is low A high concentration of nucleic acid can be achieved with a small amount of material; 2. The operation is simple and fast The whole operation process is basically divided into five steps (cracking, combining, washing, drying and eluting), the whole process does not need centrifugal operation, and most of the processes can be completed within 30 to 60 minutes; 3. The quality is stable and reliable The binding capacity of the free magnetic beads and the nucleic acid is larger, the purity of the nucleic acid is higher due to the specific binding, and the recovery amount of the nucleic acid can be adjusted by controlling the surface groups of the magnetic beads; 4. Fully automated operation The nucleic acid extraction instrument can realize automatic and high-throughput operation, and can realize the extraction of dozens or even hundreds of samples by starting with one key; 5. Safe, non-toxic and harmless The reagent does not contain toxic chemical reagents such as phenol and chloroform, which fully conforms to the modern environmental protection concept. Precautions for Nucleic Acid Extraction by Magnetic Bead Method The use of biological magnetic beads to extract nucleic acid is still a relatively new method of nucleic acid extraction in China. Compared with the traditional isoamyl alcohol extraction method and centrifugal column kit method,50l rotovap, this method is still inadvertently understood by many people. In the process of using magnetic beads to purify nucleic acid, there are also some misunderstandings. 1. Mistake one : The more magnetic beads you use, the better the extraction Many teachers like to increase the amount of magnetic beads when the extraction effect is not good, thinking that more magnetic beads can absorb more nucleic acid, which has to be said to be undesirable. The main characteristic of magnetic beads is that they can be dispersed in liquid, and can also be separated from liquid in solid state under the action of external magnetic field. In any reagent system, the ratio of magnetic beads to liquid should have a certain threshold. If the ratio exceeds a certain threshold, too many magnetic beads will lose their dispersion characteristics because they can not be uniformly dispersed in liquid. The efficiency of the contact between the nucleic acid magnetic beads and the liquid cannot be sufficiently increased during the washing process. Excessive magnetic beads will also adsorb more impurities, which has a great impact on the effect of impurity removal. Even sometimes, too many magnetic beads will adsorb protease, lysozyme and other functional components that play a major role in the liquid system, resulting in low efficiency of the whole kit. In many cases, when the extraction effect is not good, reducing the use of magnetic beads is the best way to improve the extraction effect. Usually, the amount of reference magnetic beads given by the magnetic bead method kit is slightly excessive, so it is not necessary to increase the amount of magnetic beads to improve the adsorption efficiency. However, if it is determined that the extraction effect is not good due to insufficient amount of magnetic beads, it is possible to improve the extraction effect by increasing the dosage of magnetic beads within a certain range. Taking GNT-02 series magnetic beads as an example, when extracting constant samples (plant tissues, whole blood, etc.),rotovap distillation, the dosage is usually 10 UL/time; when extracting trace samples (such as serum free DNA, oral swabs, etc.), the dosage of magnetic beads is 15 ~ 20 UL/time. If you need to exceed this usage, you need to communicate with the technical engineer. 2. Mistake two The more reagents are used, the better the extraction effect is. The cracking effect is not good? Add more lysis solution. Washing effect is not good? Add more washing up liquid. This is the inertial thinking of many customers in the use of kits. However, for the magnetic bead method, every increase in the volume of a part of the liquid reduces the collision probability of more magnetic beads, and reducing the collision probability of magnetic beads will lead to a significant decline in the adsorption rate. Therefore, in many cases, although increasing the amount of lysis solution and washing solution can indeed play a role in enhancing lysis and washing, the core of magnetic bead extraction is the efficiency of magnetic bead adsorption of nucleic acid, which can not guarantee the efficiency of nucleic acid extraction, so simply increasing the amount of reagents used to improve the extraction effect may not be completely effective. For GNT-B02 whole blood genomic DNA kit, the amount of lysis solution should not exceed 400ul/time, and the amount of washing solution should not exceed 500ul/time. If an amplification system is required, the amount of magnetic beads and sample should be increased accordingly, and this amplification is not necessarily proportional. 3. Mistake three The more times of washing, the better the extraction effect. When there are too many impurities in the extracted nucleic acid, users will consider washing several times to obtain more pure nucleic acid. Increasing the number of times of washing is indeed conducive to purifying nucleic acid, but considering that each washing will lose a certain amount of nucleic acid and increase the possibility of nucleic acid cleavage and hydrolysis, it is generally appropriate to control the number of times of washing at 2 to 4 times. For GNT series kit, the washing times of single purification kit is 2 times, the washing times of plant and animal kit is 3 times, and the washing time of blood kit is 3-4 times. 4. Mistake four : The more samples taken, the better the extraction When the sample is not fresh enough or the nucleic acid content itself is very small, the nucleic acid extraction effect is often not good, many teachers will use the way of taking more samples to increase the amount of nucleic acid extraction. However, simply increasing the sample volume may sometimes introduce too many impurities, which will exceed the cracking capacity of the cracking solution and reduce the extraction efficiency. Therefore, it is not recommended to simply increase the sample volume to achieve the purpose of increasing the extraction volume. If the extraction volume is too low due to insufficient sample size, it is recommended to start the extraction after enrichment or concentration in the pretreatment. Or increasing the completeness of the cleavage to expose more nucleic acids is also a solution. 5. Mistake five If a certain kind of magnetic bead is good, it should work well in all tests. There are many kinds of magnetic beads, such as different particle size, different dispersion, different magnetic response time, different coating base matrix, different outer modified functional groups, different coating density and different arm length of functional groups, which will lead to different characteristics of magnetic beads. Therefore, different magnetic beads are suitable for different experiments and systems. Just like the same reagent for nucleic acid extraction, the formula is not exactly the same, cbd centrifugal extractor ,jacketed glass reactor, and the properties of magnetic beads used for nucleic acid extraction are not exactly the same. Some magnetic beads show higher adsorption efficiency in the extraction of normal nucleic acid, and some magnetic beads are more suitable for the extraction of trace nucleic acid. Some magnetic beads are suitable for acidic series of reagent systems, and some magnetic beads are suitable for alkaline series of reagent systems. Some magnetic beads have good magnetic response but fast sedimentation speed, which are more suitable for magnetic rod automatic extractors; some magnetic beads have slow sedimentation speed but long magnetic response time, which are more suitable for pipetting automatic extractors. Few magnetic beads can be applied to all experimental situations, except for the fixed kit, in most cases, the magnetic beads and reagent system need to be adjusted for a certain period of time. 6. Mistake six : Compared with a certain kit, the effect is not good, but the magnetic beads are not good. In the process of screening magnetic beads, many customers simply replace the same amount of magnetic beads under the mature reagent system to compare the effect of magnetic beads. In this way, it is easy to draw the conclusion that the effect of a certain magnetic bead is not good, but in fact, because the suitable system and dosage of different magnetic beads are different, it often needs to be adjusted to obtain better extraction effect. Nucleic acid extraction and purification core parameter Extraction principle, mechanical principle, extraction time, sample handling capacity, sample tube capacity, Tip head handling capacity, number of Tip heads, interface, instrument weight, instrument size Application field of nucleic acid extraction and purification In almost every laboratory, the separation and purification work related to biomolecules is very important and indispensable. However, it is still very difficult to purify multiple samples, not only need to choose the appropriate purification technology, but also the workload is particularly large, it is difficult to meet the current rapid development of high-throughput sample extraction and purification needs. TIANLONG NP968 magnetic bead extraction and purification system uses magnetic bead technology, which can operate 1-32 samples at the same time, and can be widely used in genomics, disease control and medical treatment, food safety, forensic identification and other fields. 1. Genomics The Tianlong NP968 magnetic bead extraction and purification system is very suitable for genomics research. No matter the source of the extracted sample is microorganism, animal, plant or virus, the TIANLONG whole blood genome DNA extraction kit, the white blood cell layer whole blood genome extraction kit and the animal tissue/cell genome DNA extraction kit which are specially optimized for the TIANLONG NP968 magnetic bead extraction and purification system are combined, DNA or RNA with sufficient quantity and purity can be rapidly purified. High-quality nucleic acids can meet the needs of various downstream applications (such as PCR/Real-time PCR, gene chip, Southern blot, Northern blot). 2、CDC Based on the rapid and high-throughput technology of Tianlong NP968 magnetic bead extraction and purification, it can be used to solve the rapid automatic disease surveillance system of 2009 H1N1 flu virus (Influenza A virus subtype H1N1), children's hand-foot-mouth disease, measles virus and so on, and improve the response ability to major epidemics. The TIANLONG virus DNA/RNA extraction kit can be efficiently used for extracting RNA from cotton swabs, serum and other samples, especially for low-copy complex samples. Enterovirus EV-71 isolate (FY04-R5-C1) with an original virus content of 7.5T CID50/100μl was diluted in a 10-fold gradient of 5 dilutions. Real-time PCR detection results of each 100 μl sample after RNA extraction by centrifugal column and TIANLONG NP968 magnetic bead extraction and purification system. (Source: Chinese Center for Disease Control and Prevention) 3. Molecular diagnosis of clinical samples The Tianlong NP968 magnetic bead extraction and purification system can rapidly process clinical samples with high throughput, and the extracted nucleic acid can be used for subsequent molecular diagnosis. Tianlong NP968 combined with Tianlong FFPE tissue genomic DNA extraction kit is also suitable for formalin-fixed paraffin-embedded (formalin-Fixed and Parrffin-Embedded, FFPE) tissue samples. 4. Animal husbandry and veterinary medicine Can efficiently and sensitively extract avian influenza virus, Newcastle disease virus, Classical swine fever virus, CSFV), bovine viral diarrhea virus (Bovine viral diarrhea virus), Coxiella burnetii, etc. 5. Application of forensic medicine For forensic work, the efficiency and stability of nucleic acid extraction are very important. Tianlong NP968 magnetic bead extraction and purification system can be used with special forensic sample nucleic acid extraction magnetic bead reagent to purify high-quality DNA from different sources including cigarette butts, hair roots, cartilage, nails, blood stains and other materials, with a maximum processing capacity of 32 samples per time. Frequently asked questions about nucleic acid extraction and purification Q1: After adding Buffer 3 to the extracted plasmid, what is the effect of increasing the centrifugation speed and time on the extracted product? A1: After the addition of Buffer 3, the increase of Lixin speed and centrifugation time is indeed conducive to more compact adherence of the precipitate, but at room temperature If the centrifugation time is too long, the temperature of the liquid will rise and the plasmid will be degraded. In general, it can be centrifuged at 4 ℃ and 12000 rpm 10min, or centrifuging at 12000 rpm for 3-4min at normal temperature. Q2: During the recovery of agarose gel, if the recovered DNA fragment is too short, how to deal with it? A2: If the recovered DNA fragment is less than 500 BP, in order to improve the recovery efficiency, the gel can be dissolved by adding gel solution Then, isopropyl alcohol of 1.5 times the volume of the gel piece was added, and the mixture was thoroughly inverted and mixed. Q3: When the agarose gel is recovered, how to deal with the abnormal color after the gel liquid sol? A3: After the gel solution is completely dissolved, the normal color should be light yellow. If the color changes to red or orange, adjust the pH of the solution by adding 5.mu.l of 5m NaAc (pH 5.2) to restore the color of the mixture to the normal light yellow color (if the pH is not adjusted, the recovery of DNA will be affected). Q4: How to determine the relationship between lysate volume and sample mass during genomic DNA extraction: A4: Theoretically, the larger the volume of the lysate and the less the amount of the sample, the better the quality of the extracted product. Therefore, the best ratio of the lysis solution volume to the sample mass should be explored and optimized before the experiment. The following recommended values for the amount of sample that can be lysed with only 1 ml of lysing solution are for reference only. Q5: When extracting genomic DNA, what methods can be used to improve the yield of the product? A5-1: Properly increase the ratio of lysis solution volume to sample mass. A5-2: The eluent is preheated to about 65 ℃ in advance. At the same time, the eluent should be carefully added to the middle part of the adsorption membrane when it is loaded on the column, and ensure that the liquid completely covers the membrane. A5-3: If the volume of the mixed solution after sample lysis is too large when it is loaded on the column, it needs to be loaded on the column in several times, and ensure that the amount of sample loaded each time does not exceed 700 μl. A5-4: Properly extend the homogenization and lysis time of the tissue. Q 6: The reason for the high A260/A280 ratio of the extracted product? A6: The A260/A280 ratio of the purified DNA is between 1.8 and 2.0. If there is a large amount of residual RNA in the sample, the A260/A280 ratio will be higher, and RNase A needs to be added during the extraction process (if RNase A is added during the extraction process according to the instructions, The possibility of reduced RNase activity is considered). Q7: Precautions for sample handling prior to blood genomic DNA extraction? A7-1: Human blood contains a large number of enzyme inhibitors that may interfere with downstream DNA analysis, as well as common anticoagulants such as interferon and EDTA. Therefore, there is a need for a method for isolating DNA from human blood that provides high quality DNA free of contaminants and enzyme inhibitors. A7-2: The red blood cells of mammals do not contain nucleic acid, but the content of red blood cells in the blood of healthy mammals is nearly 1000 times higher than that of white blood cells containing nucleic acid (including lymphocytes, monocytes, granulocytes, etc.). Therefore, removing red blood cells before extracting genomic DNA can improve the yield of DNA. The recommended method is as follows: A7-2-1: Selective lysis of red blood cells: Under the condition of hypotonic buffer solution, red blood cells will undergo early hypotonic shock and rapid rupture. A7-2-2: Ficoll density gradient centrifugation to recover monocytes (lymphocytes and monocytes) and remove erythrocytes (this method also removes granulocytes). A7-2-3: The whole blood is centrifuged at 3300 G for 10 min at room temperature. After centrifugation, it will be divided into three layers: the supernatant layer is plasmid, the middle layer is white blood cell layer, and the bottom layer contains concentrated red blood cells. A7-3: The red blood cells of birds, fish, and frogs contain nucleic acids, so they do not need to be processed. A7-4: Blood samples (including blood samples that have been treated to remove red blood cells) that can be efficiently lysed with a lysing solution and protease or proteinase K. A7-5: In addition to animal genomic DNA, viral and bacterial DNA can also be isolated from blood. Q8: When extracting total RNA, how to determine the relationship between lysate and sample size? A8: Theoretically, the greater the volume of the lysate and the less the amount of sample, the better the quality of the extracted product. Therefore, the best ratio of the lysis solution volume to the sample mass should be explored and optimized before the experiment. The following recommended values for the amount of sample that can be lysed with only 1 ml of lysing solution are for reference only. Q 9: How is the sample treated for total RNA extraction? A9-1: Theoretically, the fresher the sample, the better the quality of total RNA extracted. If timely extraction is not possible, samples need to be frozen quickly with liquid nitrogen and then stored at -80 ° C at all times. Repeated freezing and thawing of samples have a great impact on the quality of extracted RNA. A9-2: Disruption: Complete disruption of tissue structures, cell walls, and plasma membranes is absolutely necessary to release all of the RNA in the sample. Different samples require different methods to achieve complete fragmentation. Incomplete fragmentation results in a significant reduction in RNA yield. A9-3: Homogenate: Homogenate is necessary to reduce the viscosity of the cell lysate after disruption. The homogenate is capable of cleaving high molecular weight genomic DNA and other high molecular weight cellular components to yield a homogeneous lysate. Incomplete homogenization will lead to the decrease of RNA binding efficiency and significantly reduce the yield. A9-4: Recommendations for different sample handling methods Q10: How to quantify RNA after extraction? A10-1: When testing RNA samples, it is necessary to ensure that RNase is removed from the cuvette, especially if it is still necessary to recover the RNA after the assay (the procedure can be followed by washing with 0.1 M NaOH, 1 mM EDTA, and water without RNa se in that order). A10-2: When measuring the absorbance value of A260 with a spectrophotometric timer using a UV-transparent plastic cuvette, the reading should be between 0.15 and 1.0. At the wavelength of A260, the absorbance value of 1 unit corresponds to an RNA concentration of 44 μg/ml (this correlation is only valid for detection under neutral pH conditions. Therefore, if RNA samples are to be diluted, a low salt buffer with a neutral pH of 10 mM Tris HCl, e.g., pH 7.0, should be used.) A10-3: example of RNA quantification RNA sample volume = 100 μl Dilution = 10 μl RNA sample + 490 μl 10 mM Tris HCl, pH 7.0 (dilution factor 1:50) Measure the absorbance of the diluted sample using a 1 ml RNase-free cuvette A260=0.2 RNA concentration = 44 μg/ml × A260 × dilution factor = 44μg/ml × 0.2× 50 =440μg/ml Article Source: Instrument and Equipment Test Special statement: The publication of this article is only for the purpose of disseminating information, and does not represent the views of this public account; if other media, websites or individuals reprint and use from this public account, please apply to the original author, and bear the copyright and other legal responsibilities. Instrument plus | dry goods finishing: chromatographic column use,winterization filtration, maintenance and repair, all for you to organize together ~ do not collect it? Instrument Plus | News and Information: Southern Hemisphere Aerosol Content Breaks Record, Air Pollution Needs Urgent Attention! 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