Nucleic acid extraction experience summary

Nucleic Acid

The biological macromolecules formed by the polymerization of nucleotide monomers are the most basic and important components of biological cells. It is generally believed that biological evolution begins with nucleic acid, because among all living substances, only nucleic acid can self-replicate. Nucleic acid is known today as a repository and transmitter of biological genetic information. The blueprint of an organism is encoded in its nucleic acid molecules. Nucleic acid was discovered in leukocytes in pus by F.Miescher in 1869. He called it a nuclide at the time. After R.Altmann recognized its acidity in 1889, he named it nucleic acid.

Classification and function of nucleic acids

Nucleic acids are divided into two categories: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). These two types of nucleic acids have some common structural features, but different biological functions.

DNA stores genetic information and replicates during cell division, so that each daughter cell receives DNA with the same structure and information content as the parent cell; RNA mainly plays a role in protein synthesis and is responsible for converting the genetic information of DNA into the amino acid sequence of a specific protein .

The basic structural unit of nucleic acid is nucleotide, which contains three components: nitrogenous base, pentose sugar and phosphate. Bases and pentose sugars form nucleosides, and the phosphate esters of nucleosides are nucleotides. The pentose sugar in DNA and RNA is different. The pentose sugar in RNA is D-ribose; DNA does not contain ribose but D-2-deoxyribose (the hydroxyl group on the 2-carbon atom in ribose is replaced by hydrogen). Nucleic acids are classified according to the types of pentose sugars in them, and the bases of DNA and RNA are also different.

Physicochemical properties of nucleic acids

Pure RNA and nucleotides are white powders or crystals, while DNA is white asbestos-like fibers. Except for inosinic acid and guanylic acid, which have umami taste, nucleic acids and nucleotides are mostly sour. DNA, RNA and nucleotides are polar compounds, generally soluble in water, insoluble in organic solvents such as ethanol and chloroform, their sodium salts are more soluble in water than free nucleic acids, and the solubility of RNA sodium salts in water can reach 40g/ L, DNA sodium salt is 10g/L in water, and it is a viscous colloidal solution. In acidic solution, DNA and RNA are easily hydrolyzed and stable in neutral or weak alkaline solution. DNA in its native state is present in the nucleus in the form of deoxyribonucleoprotein (DNP). **When extracting DNA from cells, first extract DNP, then remove P, and then remove sugar, RNA and inorganic ions in cells to separate DNA from it.

Cell Lysis:

The principle of lysis In the nucleic acid extraction process, cell lysis is very important.

Classical lysates almost all contain detergents (such as SDS, Triton X-100, NP-40, Tween 20, etc.) and salts (such as Tris, EDTA, NaCl, etc.). The role of salt, in addition to providing a suitable lysis environment (such as Tris), also includes inhibiting nucleic acid damage (such as EDTA) by nucleases in the sample during the lysis process, maintaining the stability of nucleic acid structure (such as NaCl), etc. Detergent is to denature the protein, destroy the membrane structure and untie the protein connected with the nucleic acid, so that the nucleic acid is free in the lysis system. Protease may also be added to the cleavage system, and the protein is digested into small fragments by protease, which promotes the separation of nucleic acid and protein, and also facilitates subsequent purification operations and obtains purer nucleic acid. There are also directly lysed with high concentrations of protein denaturants (such as GIT, GuHCl, etc.), which has become the mainstream of RNA extraction, but not the mainstream of genomic DNA extraction.

Cell lysis methods Bacterial cell disruption methods include the following:

1) Mechanical methods: ultrasonic treatment, grinding, and homogenization. Regarding the ultrasonic treatment method, it is necessary to set the ultrasonic time and the gap time. Generally, the ultrasonic time does not exceed 5 seconds, and the gap time is preferably greater than the ultrasonic time. 2) Chemical reagent method: The cells are treated with a solution containing SDS or CTAB. Under a certain pH environment and denaturation conditions, the cells are ruptured, the protein is denatured and precipitated, and the nucleic acid is released into the aqueous phase. The pH environment is controlled by the addition of strong alkali (NaOH). ) or buffers (TE, STE, etc.), surfactants or strong ionic agents can cause cell lysis, protein and polysaccharide precipitation, and some metal ion chelators (EDTA, etc.) in the buffer can chelate nuclease activity. The necessary metal ions Mg2+ and Ca2+ can inhibit the activity of nucleases and protect nucleic acids from being degraded. 3) Repeated freeze-thaw method: freeze the cells below -20 degrees, thaw them at room temperature, and repeat several times. Due to the formation of ice particles in the cells and the increase of the salt concentration of the remaining cell fluid, the swelling will be caused, and the cell structure will be broken. Personal experience in general, 37 ℃, 3min, liquid nitrogen 3min, repeated three times. 4) Enzymatic hydrolysis method: adding lysozyme or helicase, proteinase K, etc., can break the cell wall and release nucleic acid. Proteases can also degrade proteins bound to nucleic acids and promote the separation of nucleic acids. Among them, lysozyme can catalyze the hydrolysis of β-(1,4) bonds between the proteoglycan N-acetylglucosamine and N-acetylmuramic acid residues in bacterial cell walls. Proteinase K can catalyze the hydrolysis of a variety of polypeptide bonds, and it retains its enzymatic activity at 65 °C and in the presence of EDTA, urea (1-4 mol/L) and detergent (0.5 % SDS or 1 % Triton X-100). , which is beneficial to improve the extraction efficiency of high molecular weight nucleic acid. In practical work, enzyme action, mechanical action and chemical action are often used in combination. Which method or methods to choose can be determined according to the cell type, the type of nucleic acid to be isolated, and the purpose of subsequent experiments.

Evaluation of cracking methods

Protease-containing cleavage methods can be considered the first choice for the extraction of genomic DNA. Cleavage includes free membrane proteins and free proteins linked to genomic DNA. The role of protease is to make the protein smaller, so it has a huge promotion effect on the free protein; at the same time, the huge genomic DNA is easy to "entangle" the macromolecules, and after the protein is digested by the protease, it is not easy. Being "entangled" by genomic DNA is conducive to the removal of proteins during purification operations, resulting in higher purity of the final genomic DNA. Another idea is that if the genomic DNA is "entangled" with the protein, there are two possibilities during the purification process: if the characteristics of the genomic DNA are dominant, it will be retained in the form of DNA during purification, resulting in the residue of the protein; If the properties of the protein predominate, it is removed in the form of protein during purification, resulting in loss of DNA. Of course, the detergent lysis method still has advantages in the extraction of cellular genomic DNA, especially when the yield and purity requirements are not the highest, and economy and simplicity of operation are important. Controlling the ratio of lysate ****/**** sample is the key to the success of this method. This method combined with high-salt precipitation can achieve the simplest operation, but the stability of purity and yield may be worse than that of extraction with PC.

The lysis method of high concentration protein denaturant (such as GIT, GuHCl, etc.) is the first choice for RNA extraction. For the extraction of total RNA, the most important thing is to quickly lyse the cell membrane. As for the cleavage of the protein connected to the genomic DNA and the problem of "entanglement" between the genome and the protein, it will not have a great impact on the subsequent purification. consider. High concentrations of protein denaturants can rapidly destroy cell membranes, thereby rapidly inhibiting intracellular RNases, thereby ensuring RNA integrity. Extraction of RNA from the vast majority of samples, except for a very small number of samples that are not suitable for this method - mainly plants - can be based on high concentrations of protein denaturants. Of course for some samples, such as muscle, even for RNA extraction, the use of a protease-containing lysis buffer (or protease digestion of the protein at some point in the procedure) is strongly recommended because the protein in these samples is very difficult to remove . This method is the basis for obtaining maximum yield and purity.

The CTAB-containing lysate has almost become the preferred lysis method for the extraction of genomic DNA from polysaccharide-rich samples such as bacteria and plants. The success of this method is related to two factors: the quality of the CTAB and the thoroughness of the washing. The quality of CTAB has a great influence on the cracking efficiency, and it seems unclear why, because even CTAB of the same purity produced by the same company, with different batch numbers, may have very different effects. It is more difficult to remove CTAB by washing than other salts, and at the same time, a small amount of CTAB residue will also have a huge impact on the enzyme activity, so the thorough washing is also the key to the success of this method. For the pyrolysis temperature, use 65C; but if you find that the degradation is severe or the yield is too low, you can try the relatively low temperature region of 37C – 45C.

SDS alkaline lysis method is the preferred lysis method for plasmid extraction, with the characteristics of rapidity, high yield and almost no genomic DNA contamination. Controlling the ratio of lysate/cell and mild operation are the keys to the success of this method. The protein precipitation efficiency is better at 4C, so the quality can be improved by adding solution III and then standing at 4°C for a period of time and centrifuging at 4°C to remove the protein. This method does not necessarily require the use of PC purification, but in combination with PC purification, very high-purity plasmids can be obtained. Removal of RNA can be achieved by adding RNase A (100ug/ml) to solution I or RNase A (25ug/ml) to the final lysate. The general feeling is that with RNase A in solution I, there is less residual RNA. However, classical precipitation is almost impossible to completely remove RNA residues. Additionally, this method can be problematic for large plasmids (above 50 kb).

The simple cleavage method of PCR template is also a widely used method. The feature of this method is that no purification is required, and the lysate can be directly taken for PCR after the sample is lysed, which is very fast. Because of the lack of purification, the proportion of false negatives (that is, positives that are not amplified) is also relatively high. The simplest method is the repeated freeze-thaw method, which is simple and quick, does not require any chemical reagents, freeze-thaw centrifugation, and PCR detection. If the lysis method is used, the simplest lysis solution is water, and the more complex ones will contain some things that will not inhibit the subsequent PCR reaction, but can improve the lysis efficiency, and may even partially eliminate the impurities in the sample that inhibit the PCR reaction. Such as Triton X-100, formamide and so on. A little more complex will contain media such as Chelex 100 that can absorb some of the impurities. The operation is also very simple, and the temperature change is often used to achieve sample cracking, such as boiling, or multiple cycles of high temperature and low temperature. This method is most suitable for finding positive samples from a large number of samples, but it is not suitable for determining whether a sample is positive or negative. Reducing the amount of sample used can improve the positive rate, because the reduction of the sample amount also means that the amount of PCR inhibitor is reduced.

The principle of the amount of lysis solution is to ensure that the sample can be completely lysed, and at the same time, the concentration of nucleic acid in the lysis system is moderate. If the concentration is too low, the precipitation efficiency will be low and the yield will be affected; if the concentration is too high, the process of removing impurities will be complicated and incomplete, resulting in a decrease in purity. Also, keep in mind that the amount of lysate used is based on the protein content of the sample, not the nucleic acid content.

Nucleic acid purification

As far as I know, the nucleic acid purification technologies widely used in scientific research can be divided into two categories: those that use media and those that do not use media, those that use media to separate nucleic acids from all other impurities at one time; those that do not use media. , must first separate nucleic acids and salts from macromolecular impurities, and then separate nucleic acids from salts by precipitating nucleic acids (except for PEG precipitation and LiCl precipitation).

1) The classic purification technique using phenol/chloroform extraction: after cell lysis, centrifuge the nucleic acid-containing aqueous phase, and add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1 volume) mixture. Depending on the application, the two phases are mixed by vortex (suitable for the isolation of small molecular weight nucleic acids) or simply by inversion (suitable for the isolation of high molecular weight nucleic acids) and then centrifuged. Hydrophobic proteins are partitioned into the organic phase, while nucleic acids are retained in the upper aqueous phase. Phenol is an organic solvent, which is pre-saturated with STE buffer, because unsaturated phenol will absorb the aqueous phase and take away part of the nucleic acid. Phenol is also easy to oxidize and turn yellow, and oxidized phenol can cause phosphodiester bonds in nucleic acid chains to break or cross-link nucleic acid chains; therefore, a special substance should be added when preparing phenol saturated solution to prevent phenol oxidation. Chloroform removes fat and denatures more protein, thereby increasing extraction efficiency. Isoamyl alcohol reduces the generation of air bubbles during operation. Nucleic acid salts can be precipitated by some organic solvents, which can concentrate nucleic acids, change the type of nucleic acid solubilization buffer and remove certain impurity molecules. A typical example is ethanol precipitation after extraction with phenol and chloroform, adding NaAc or KAc with pH 5.0 to 5.5 and a final concentration of 0.3M to the nucleic acid-containing aqueous phase, the sodium ions will neutralize the nucleic acid phosphate backbone. The negative charge promotes the hydrophobic renaturation of nucleic acids in an acidic environment. Then add 2-2.5 times the volume of ethanol, after a certain period of incubation, the nucleic acid can be effectively precipitated. Other organic solvents (isopropanol, polyethylene glycol (PEG), etc.) and salts (10.0mol/L ammonium acetate, 8.0mol/L lithium chloride, magnesium chloride and low concentration of zinc chloride, etc. ) is also used for the precipitation of nucleic acids.

2) Purification technology using ion exchange medium: the lysate is passed through the column, and the nucleic acid is linked to the ion exchange medium; after washing to remove residual impurities, the nucleic acid is eluted from the medium with a high-salt buffer. After standard ethanol/isopropanol precipitation, ethanol washing, drying and other operations, pure nucleic acid is obtained, which is dissolved in a suitable buffer. 3) Purification technology using adsorption medium: the lysate is passed through the column, and the nucleic acid is selectively adsorbed by the adsorption medium; after washing to remove residual impurities, the nucleic acid is eluted from the medium with water or a suitable low-salt buffer, and the nucleic acid can be directly used for subsequent experiments. 4) Density gradient centrifugation: Density gradient centrifugation is also used for the separation and analysis of nucleic acids. Double-stranded DNA, single-stranded DNA, RNA and protein have different densities, so pure sample bands of different densities can be formed by density gradient centrifugation. This method is suitable for the preparation of a large number of nucleic acid samples. Ingot gradient equilibrium centrifugation is considered the method of choice for purification of large quantities of plasmid DNA. Cesium chloride is the standard medium for nucleic acid density gradient centrifugation. Ethidium bromide in the gradient solution is combined with nucleic acid. The nucleic acid zone formed after centrifugation is irradiated by ultraviolet light to generate fluorescence and detected. The cesium chloride is removed by dialysis or ethanol precipitation to obtain purified nucleic acids.

Evaluation of purification methods

PC (P is the abbreviation of English phenol, C is the abbreviation of English chloroform) extraction/alcohol precipitation method is a timeless method. Stable, reliable, economical and convenient. PC extraction can completely remove proteins, and alcohol precipitation can remove salts. For general clean samples (impurities are proteins), this method can completely obtain high-quality nucleic acids. Although a portion of the nucleic acid is lost with each PC extraction (because it is impossible to pipette the entire aqueous phase), and the alcohol precipitation of low-concentration nucleic acid is inefficient, these problems can be solved or reduced by adjustment of the operation. The biggest problem with this method is that it is not suitable for large-scale extraction. PC extraction is a very effective means of removing proteins. Phenol can denature the protein, and the denatured protein is precipitated from the aqueous phase, in the phenol or between the phenol/water phase. The key to PC extraction is to mix thoroughly, and to use enough amount. Mix thoroughly to ensure adequate contact between phenol and protein to fully denature the protein. Many people are always concerned that the intensity of mixing will damage nucleic acids, especially genomic DNA, but it is not necessary to be so careful. Vigorous mixing operation will partially disrupt the genomic DNA of macromolecules, but the disruption will not be so strong that the DNA becomes small fragments within 10kb. After shaking and mixing vigorously by hand, most of the genomic DNA fragments will be larger than 20kb. This size, except for some special requirements, is completely suitable for PCR and enzyme digestion. If the required fragments are very large, such as for library construction, vigorous mixing methods cannot be used, but gentle back-and-forth inversions – the key here is that the proportion of lysate is large enough that the system is not too viscous . The amount should be sufficient, because the removal of protein by phenol has a certain degree of saturation. If the saturation is exceeded, the protein in the cleavage system will not be removed once, and must be completely removed by multiple extractions. In addition, the disadvantage of a too viscous system is that the protein is difficult to completely remove, and the genomic DNA will be more broken, so pay attention to the ratio of lysate to sample. Centrifugation at 4C facilitates more complete protein removal. Another use of PC extraction is that the use of acidic phenols can partially remove the characteristics of DNA and obtain RNA with very little DNA residue during RNA extraction. However, it should be reminded that some plant samples cannot be extracted with PC until some impurities are removed, otherwise the nucleic acid will be degraded.

High salt precipitation protein/alcohol precipitation method is also a very good method. Compared with the PC extraction method, this method overcomes almost all the disadvantages of PC extraction except that the stability of purity may be a little lower. The concomitant benefit of faster and easier protein removal is that it can be used for large scale extractions, the disadvantage is that the purity (protein residue) is not stable enough. Protein precipitation efficiency is better at 4C.

The medium purification method is an increasingly important method. Its biggest feature is that it is very suitable for large-scale nucleic acid extraction, and because it is less affected by human operating factors, the stability of the purity is high (although the purity is not necessarily higher than the PC purification method). Its Achilles heel is sample excess. Media can be divided into two categories, one is column type, that is, the medium is pre-packed in a column with a pass below; the other is granular (such as Glassmilk, magnetic beads, etc.). The purification operation of the granular medium is not much different from the classical alcohol precipitation. It is through several additions and pouring processes. After drying, the purified nucleic acid can be obtained by dissolving. Although the operation of column purification also has the process of adding liquid and pouring liquid, because the added liquid will enter another centrifuge tube after centrifugation, which is completely separated from the column containing nucleic acid, so the washing is more thorough and the operation is more labor-saving ( Don't worry about pouring out the nucleic acid, or the residue of the liquid). However, the cost of the media purification method is the highest.

Alcohol precipitation

1) The principle and purpose of precipitation: The purpose is to precipitate nucleic acid from the lysis system, so as to achieve the separation of nucleic acid and other impurities - mainly salts. As far as nucleic acid is concerned, standard alcohol precipitation requires a certain amount of salt and a certain proportion of alcohol, but this by no means means that these salts are indispensable or that the proportion of alcohol cannot be changed. In practice, it is not difficult to find that when the nucleic acid concentration in the lysis system reaches a certain level, even if the system does not contain the salt recommended in the textbook, the nucleic acid can be precipitated by using alcohol alone; Nucleic acids can be precipitated (of course, yields may be reduced). The point of knowing this is: don't be superstitious about the uniqueness of standard methods; instead, when you run into problems with standard methods—primarily purity issues, you can simply adjust the precipitation conditions to improve. The most valuable reference is a precipitation scheme: replacing pure isopropanol with half isopropanol and half high-salt solution, which can greatly reduce polysaccharide residues. Another problem is that it must be firmly believed that the alcohol precipitation process of nucleic acid is also the precipitation process of other impurities; although adjusting the conditions of alcohol precipitation will reduce the yield of nucleic acid, it can greatly improve the purity.

The choice of precipitant: using isopropanol or ethanol for alcohol precipitation, we did not find that the two have a great impact on the quality. The nucleic acid precipitated by isopropanol is relatively compact, the wall is tight, and the color is not very white; the nucleic acid precipitated by ethanol is relatively fluffy, easy to move from the wall, and the color is relatively white. This is the phenomenon that a small amount of nucleic acid is precipitated with isopropanol, and a large amount of nucleic acid is precipitated with ethanol. As for the statement that isopropanol precipitation is easier to precipitate salt, we have never encountered it, and I think the reason for this phenomenon is that the washing is not thorough. Of course, don't forget that the biggest advantage of isopropanol precipitation is its small size, which allows most of the small extraction operations to be completed in a 1.5ml centrifuge tube. However, because the precipitate is very compact, the central part of the precipitate is not easy to be washed during washing. Therefore, the key to washing nucleic acid precipitated by isopropanol is: the precipitate must be suspended, and it must be placed for a period of time to make the precipitate finally fluffy. white. If you wash it again, the quality will never be a problem. Of course, the reagents used for precipitation, not only ethanol and isopropanol, but also PEG, LiCl, and CTAB can be used for nucleic acid precipitation. Although they are far from the high frequency of alcohol precipitation, they have their own characteristics. LiCl precipitates RNA to remove DNA, and CTAB precipitates nucleic acids from polysaccharide-containing lysis systems. PEG is a convenient means of precipitating viral particles. 3) Selection of precipitation temperature: If the initial sample for nucleic acid extraction contains many impurities, in principle, low temperature precipitation should not be used. Low temperature precipitation can improve the precipitation efficiency: when the nucleic acid concentration is very low, the effect is obvious; when the nucleic acid concentration is relatively high, the effect is not obvious, but it will lead to a great increase in impurities.

Nucleic acid washing

1) Washing principle and purpose: Washing is also very important for the entire extraction process. For washing, the precipitate must be suspended first; the second is to control a certain time, especially when the nucleic acid precipitate is relatively large (the nucleic acid precipitate will eventually be fluffy); the third is a small amount of time; the fourth is to remove the supernatant. thorough. The operation in most of the data is basically "discard the supernatant and place it on the absorbent paper for a while". This statement means that for a good quality centrifuge tube, if the centrifuge tube is siliconized, because the liquid hardly hangs on the wall, Therefore, the supernatant can be removed very thoroughly; for a good tube, even if it has not been siliconized, the residual amount is very small, and there is no problem; for a poor tube, the liquid hanging on the wall is very considerable, and the residual amount is so large that it will affect the subsequent experiments. . A good way to avoid liquid residues is to pour off the liquid and then briefly centrifuge it to remove the residue with a pipette. It is also necessary to keep in mind that the residual liquid contains impurities from the previous operation, and the residual amount is related to the degree of mixing and the size of the nucleic acid precipitate. Ethanol is removed during volatilization, and impurities will not be removed by volatilization. In this step, don't shake it with your hands, it is easy to get rid of the nucleic acid. In addition, the size of the nucleic acid pellet and the lysis capacity of the lysate also determine the intensity of washing. The larger the precipitation, the stronger the lysing ability of the lysing solution, and the more thorough the washing is: the storage time is relatively longer, and the number of washings should also be increased. Of course, the choice of ethanol concentration is also very important. Generally, 75% ethanol at room temperature must be used for washing.

Nucleic acid solubilization and preservation

1) Nucleic acid dissolution: The purified nucleic acid, in which RNA is mainly dissolved in water, and DNA is mostly dissolved in weakly alkaline Tris or TE. The classical DNA solubilization method advocates the use of TE solubilization. The reason is that some people think that EDTA can reduce the risk of DNA being degraded by DNase that may remain; if the operation process is properly controlled, the residue of DNase is almost negligible, and Tris can be used directly. Alternatively water (pH close to 7) dissolves the DNA.

2) Nucleic acid preservation: Basically, the stability of nucleic acid in preservation is inversely proportional to temperature and proportional to concentration. Generally, we choose, -20 ℃ or 70 ℃ storage stability. If the temperature is not suitable, the nucleic acid will degrade or disappear during storage. The first reason is the enzymatic hydrolysis caused by the residual enzyme, and the second reason is the hydrolysis caused by the unsuitable pH value of the nucleic acid solution (RNA is more stable in weak acidity, while DNA is more stable). More suitable in weak alkaline). Another thing that is not taken seriously is the impact of EP **** tubes on nucleic acids. First of all, it must be firmly believed that nucleic acid will definitely react with the contact surface of the container in which it is placed to reach a certain equilibrium. The material of EP **** tube may firstly adsorb nucleic acid, and secondly, it can induce some changes in the structure of nucleic acid, such as denaturation. When the nucleic acid concentration is relatively high, this phenomenon may not be observed; when the nucleic acid concentration is very low, it is more obvious. Adding Geletin, Glycogen, and BSA to low concentrations of nucleic acid can stabilize nucleic acid. Although it has been proved by experiments, many experimenters do not take this suggestion seriously. There are far more materials that make EP pipes now than in the past. These emerging materials may be much better than the original pure PP materials in terms of physical characteristics such as strength and transparency, but their chemical characteristics, especially their possible effects on nucleic acid stability, are far from thoroughly studied. Just as the current plasmids can be transformed to be more and more suitable for certain requirements, the negative product may be that the extracted plasmids have more and more configurations in electrophoresis: in addition to the original three band types, there may also appear denatured cc, multimeric forms of cc and so on.

Nucleic acid concentration

The most widely used nucleic acid concentration method is ethanol precipitation. In the presence of a moderate concentration of monovalent cations, after adding a certain amount of ethanol, the formed nucleic acid precipitate can be recovered by centrifugation, and even DNA or RNA as low as pg can be quantitatively recovered. The recovered nucleic acid can be redissolved in an appropriate buffer at the desired concentration. For the specific operation of nucleic acid concentration, add V/10 monovalent cationic salt storage solution 2V absolute ethanol to the small centrifuge tube containing the sample, mix well, put it in an ice-water bath for 15-30 minutes, take it out and check the balance, 0-4 degrees, 12000g, centrifuged for 10min. Aspirate and discard the supernatant, and then wash with another 70% ethanol 0.5-1ml, 12000g, 0-4 degrees centrifugation for 2min. Aspirate and discard the supernatant, drain the pellet with an oil pump or open the lid to dry, and dissolve in an appropriate volume of buffer. The choice of monovalent cation salt is mainly based on the following considerations: the use of ammonium acetate can reduce the co-precipitation of dNTPs, but ammonium acetate should be avoided when phosphorylating nucleic acids in the future, because the ammonium ion is T, and the polynucleotide kinase is strongly inhibitor. When precipitating RNA with a higher concentration of ethanol, LiCl is commonly used, because LiCl is highly soluble in ethanol and does not co-precipitate with nucleic acids. For nucleic acid samples containing SDS, NaCl should be used, and the detergent should remain soluble in 70% ethanol. DNA and RNA precipitation, mostly using sodium acetate (pH 5.2).

Nucleic acid quality detection problems

Using the extracted nucleic acid directly for subsequent experiments is the only reliable detection method; other detection methods are relative and not very reliable. At present, the methods used to detect the quality of nucleic acid before formal experiments are electrophoresis and ultraviolet spectrophotometer. Electrophoresis mainly detects the integrity and size of nucleic acid. As long as the nucleic acid is not too small or too large (beyond the range of electrophoretic separation), this method is still very reliable; electrophoresis can also be used to estimate the concentration of nucleic acid, but its accuracy is similar to that of nucleic acid. Experience is relevant; in addition, electrophoresis may also provide information on contamination by certain impurities, but is also empirical. UV spectrophotometers measure purity and nucleic acid content, however, because UV spectrophotometers cannot be guaranteed to be very accurate, and the sensitivity of the instrument is very high, the results provided are not very reliable. Generally speaking, a more reasonable judgment can be made by combining the results of UV and electrophoresis detection at the same time. But since both methods have flaws, it shouldn't be a fuss if bad results can be used in follow-up experiments and good results can't be used in follow-up experiments. About the detection of UV spectrophotometer. First of all, don't use instruments that can't choose wavelengths; use those instruments that have fixed wavelengths, which is probably the beginning of your nightmare (no information is terrible, and even more terrible is to take wrong information seriously, among which 280, 320, 230, The absorbance at 260nm represents the value of nucleic acid, background (solution turbidity), salt concentration and organic matter such as protein, respectively.). Secondly, be sure to adjust the instrument frequently; finally, remember that the readings A230:A260:A280 = 1:2 (1.8 for DNA): 1 is theoretical data, there will be some differences in actual measurement; but if the difference is too large , there is a problem, there are two debatable points: if A260/A230 > 2 is pure, if A260/A280 > 2 is nucleic acid degradation. If A260/A230 is much larger than 2.0, there must be impurities remaining (I don't know what the impurities are). If the nucleic acid is measured immediately after extraction, A260/A280 > 2.0 will never indicate nucleic acid degradation, because the nucleic acid fragments that can lead to A260/A280 > 2.0 are very small, and it is impossible to precipitate them by conventional precipitation; A more likely reason is that the A260/A280 provided by your instrument is actually the value of A262/A282 or even A264/A284. The absorbance of pure nucleic acid is the bottom at A230, the peak at A260, and the half-hill at A280. According to the characteristics that the slope of the curve is the smallest at the bottom and the top, and the slope is the largest at the half-hill slope, it is not difficult to draw the following conclusion: the readings of A230 and A260 are less affected by the accuracy of the instrument, while the A280 is more affected by the accuracy of the instrument. Regarding electrophoretic detection, the easiest way to visualize DNA in an agar gel is to stain it with the fluorescent dye ethidium bromide. The substance contains a planar group that can intercalate between stacked bases of DNA. The fixed position of this group and its close proximity to the bases cause the dye to bind to the DNA and fluoresce with a higher fluorescence yield than the free dye. solution increased. DNA absorbs UV radiation at 254nm and transfers it to the dye, while the bound dye itself absorbs light at 302nm and 366nm. In both cases, the absorbed energy can be re-emitted at 590 nm in the red-orange region of the visible spectrum. Therefore, small amounts of DNA can be detected when the gel contains free ethidium bromide.

It is recommended to first detect by electrophoresis, and then detect by UV spectrophotometer. After electrophoresis, you can see which samples cannot be used at all (such as too much degradation), as well as the approximate concentration of nucleic acid, which provides a reference for the detection of UV spectrophotometer (degradation is unnecessary to measure, the how much to take, etc.).

Removal of impurities in nucleic acids

For some experiments that require high nucleic acid purity, we need to carry out special treatment to remove polysaccharides, proteins and non-target nucleic acids. The methods are as follows:

(1) Glycogen, starch and mucopolysaccharide often remain in the extract due to their physical and chemical properties similar to those of nucleic acid. The removal methods are often as follows: (1) The content of polysaccharide in the tissue should be reduced as much as possible before taking the material. If the animal is starved for several days and then killed, the intracellular glycogen can be greatly reduced. ② Add amylase to decompose macromolecular polysaccharides into small molecules, which are gradually removed in subsequent purification steps. ③ In the presence of concentrated phosphate, extract the nucleic acid extract with 2-methoxyethanol, so that the polysaccharide is dissolved in the lower aqueous phase, and the nucleic acid is in the upper organic layer. ④ Precipitate DNA with calcium salt, then treat with potassium oxalate to form potassium salt of DNA for recovery, and then use ion exchange method to adsorb DNA to separate it from polysaccharide.

(2) Removal of proteins: Since nucleic acids exist in the form of ribosomes in cells, no matter which method is used to extract nucleic acids, proteins exist in the system to varying degrees. Therefore, protein removal is an inevitable step in nucleic acid isolation and purification. Common methods are as follows: ① Add detergent such as sodium lauryl sulfate, and this method can be used repeatedly in all stages from extraction to separation and purification. Detergents are used in combination with the chloroform method or the phenol method, and the effect is more ideal. ②Chloroform-amyl alcohol or octanol shakes the extract to extract, and the protein forms a gel at the chloroform-water interface, which is removed after centrifugation, and the nucleic acid remains in the aqueous solution. This method is often used repeatedly in separation and purification. ③Phenol aqueous solution extraction, in the presence of anionic compounds such as p-aminosalicylic acid, DNA or RNA can enter the water phase, and proteins are precipitated in the phenol layer, and then add ethanol or 2-ethoxyethanol to the water phase to precipitate RNA or DNA , the residual phenol can be removed by Sephadex G-10 or G-25.

(3) Separation of different types of nucleic acids: During the preparation of two types of nucleic acids, it often occurs that a small amount of RNA is mixed in DNA products or a small amount of DNA is mixed in RNA products. Since the structures and properties of DNA and RNA are very similar, and the molecular weights are very large, the separation of the two types of nucleic acids is a complicated and tedious step in nucleic acid purification. The methods of removing RNA from DNA are often as follows: ① RNase selectively destroys RNA, which is a more effective method, but the ribonuclease used often contains a very small amount of deoxyribonuclease, which must be removed by heat treatment in advance, and then The RNA destruction can be achieved by incubating pure ribonuclease with the sample solution at 37°C for several minutes. ②Calcium salt step-by-step precipitation: add 1/10 volume of 10% calcium chloride solution to the nucleic acid solution to make both DNA and RNA into calcium salts, and then use DNA calcium salt to form precipitation in 2/10 volume of ethanol. RNA calcium salts are separated from each other without forming a precipitate. ③ Activated carbon adsorption: Add the treated activated carbon into the solution containing 0.5-1 mg DNA per ml at 1/15-1/20 volume, 0-4. Stir at C for 1h, centrifuge at 31,000×g for 1h, and the DNA recovery rate after removing RNA can reach 94%. For the removal of DNA from RNA preparations, aqueous phenol extraction is a more effective and commonly used method. In the absence of anionic compounds, most of the DNA can be removed by repeated extraction of RNA with an equal volume of 90% phenol in water. In addition, DNA can also be destroyed by adding deoxyribonuclease treatment, or the DNA can be removed with reference to the above-mentioned DNA and RNA separation method.

(4) Separation of similar nucleic acids: ①Separation of RNA mixture: The extracted and preliminary purified RNA products contain various RNAs and some degraded RNA mixtures. For further purification, methods such as column chromatography, gradient centrifugation and countercurrent fractionation are often used. For example, the separation of tRNA and rRNA is carried out on a methylated albumin column adsorbed on the surface of diatomaceous earth, followed by elution with different gradients of sodium chloride solution, or centrifugation with a sucrose gradient (5% concentration at the top, 20% concentration at the bottom) Separate. The metabolism of mRNA is fast, with an average lifespan of 90 minutes in bacteria and about several hours to ten hours in mammals. Density gradient centrifugation and DNA-agar columns are commonly used for separation. When preparing rRNA, due to co-precipitation, mRNA is often mixed, and it can be treated with naphthalene-1,5-disulfonic acid to separate the two. The purification of various tRNAs is usually carried out under the phosphate buffer-formamide-isopropanol system by the countercurrent fractionation method, and the separation effect is better. ②Separation of DNA mixture: mainly denatured or degraded DNA and natural separation, commonly used calcium phosphate, ECTEOLA-cellulose, DEAE-cellulose and methylated albumin column chromatography, countercurrent fractionation, gradient centrifugation and other methods. A highly purified DNA product with biological activity should have the following standards: no protein and polysaccharide; no dialyzable small molecule impurities; at pH 7, the maximum UV absorption is between 257 and 261 μm, and E(P) is 6600 It does not contain RNA; the solid is fibrous, and the aqueous solution has high viscosity and flow birefringence; it has electrophoretic homogeneity and monodispersity in ultracentrifugation; it has biological activity.

Reference: From the blog of Bai Changming Science Network

The nucleic acid extraction system (referred to as "extraction system" below) produced by Jiangsu Bioperfectus Technologies Co., Ltd. (referred to as "Bioperfectus" below) is an in vitro diagnostic medical device, which integrates cutting-edge mechanical, electronic and software technologies to enable automated extraction of nucleic acids from samples. Scope of application: The equipment is intended for use with magnetic bead-based nucleic acid extraction kits for extraction and purification of nucleic acids from clinical samples. Please read this manual carefully before using this equipment, and operate it according to the precautions. Put the manual near the extraction system for reference when necessary.?

The product is intended for use with magnetic bead-based nucleic acid extraction kits for the extraction and purification of nucleic acids from clinical samples. The product should be used by doctors, nurses, or biochemistry laboratory technicians in PCR laboratories and biosafety cabinets, who should process the sample to be tested before adding it to the sample wells in the 96-well plate pre-filled with reagents for nucleic acid extraction systems, open the equipment door, place the 96-well plate and sheaths, and start the extraction system after closing the door. The nucleic acid extraction system first heats the sample for cell lysis, and then the magnetic bars and sheaths absorb the magnetic beads and move them to the sample wells. After the magnetic beads absorb the nucleic acids released from cell lysis, they are transferred to the washing wells through the magnetic bars and sheaths. Specific steps: sample processing - lysis - binding - washing - elution. Purified nucleic acids ready for use after the magnetic beads are removed The system utilizes the magnetic bead technology for nucleic acid extraction, which uses a protein denaturant as the cell lysis buffer to lyse cells of plants and animals, denature DNA-binding proteins, and release DNA. The magnetic beads are used to specifically adsorb the DNA and are washed to remove impurities such as proteins and polysaccharides. The eluent is used to elute the DNA from the magnetic beads to obtain pure DNA at a high concentration, which is ready for PCR, genetic engineering and other procedures.?

For more information please review https://www.bioperfectus.com/