1. Introduction Many methods have been proposed for the purification of plasmid DNA from bacteria. These methods contain the following three steps: Growth of bacterial culture. (1) Growth of bacterial culture Pick a single colony from the agar plate and inoculate it into the culture (there is growth in a liquid medium containing antibiotics), and then purify the plasmid from it. The purification of the plasmid is almost always the case. Many plasmid vectors used today (such as the pUC series) can replicate to very high copy numbers, but as long as the culture is grown in standard LB medium until late logarithm, the plasmid can be purified in large quantities. At this time, it is not necessary to amplify plasmid DNA reversibly. However, the longer-generation vectors (such as pBR322) cannot replicate so freely, so it is necessary to add chloramphenicol to the partially grown bacterial culture and continue culturing for several hours in order to sexually amplify the plasmid. Chloramphenicol can inhibit the host's protein synthesis, which prevents the replication of bacterial chromosomes. However, the relaxed plasmid can continue to replicate, and its copy number continues to increase within a few hours. In this way, the production of plasmids like pBR322-like plasmids from chloramphenicol-treated and untreated cultures is very different, and the former is greatly increased. For many years, the addition of chloramphenicol (g / ml) sufficient to completely inhibit protein synthesis has become a standard operation, and the amount of plasmid DNA extracted by this method has been the task for almost all tasks imagined in molecular cloning. (2) Harvest and lysis of bacteria Bacteria can be harvested by centrifugation, and bacteria can be lysed by any of a variety of methods, including non-ionic or ionic detergents, organic solvents or alkalis, and heat treatment. . Which method to choose depends on three factors: the size of the plasmid, the Enterobacter strain and the technique used to purify the plasmid DNA after lysis. Although it is impractical to propose precise lysis conditions for each combination of plasmid and host, it is still possible to select an appropriate method according to the following general guidelines to achieve satisfactory results. (3) Purification of plasmid DNA All commonly used purification methods take advantage of the two properties of relatively small plasmid DNA and covalently closed loops. For example, the separation of plasmid and chromosomal DNA by cesium chloride-ethidium bromide gradient equilibrium centrifugation depends on the amount of binding of ethidium bromide to linear and closed-loop DNA molecules. Ethidium bromide binds to DNA by intercalating between bases, which unwinds the double helix. This leads to an increase in the length of the linear DNA. As compensation, supercoiled units will be introduced into the closed-loop plasmid DNA. Finally, the degree of supercoiling is greatly increased, which prevents the continued insertion of ethidium bromide. However, linear molecules are not limited to this, and can continue to bind more dye until saturation is reached (about 1 ethidium bromide molecule per 2 base pairs). Due to the difference in the amount of dye binding, the buoyancy density of linear and closed-loop DNA in cesium chloride containing saturated ethidium bromide is also different. For many years, cesium chloride-ethidium bromide gradient equilibrium centrifugation has become the method of choice for preparing large amounts of plasmid DNA. However, this process is expensive and time-consuming, and many alternative methods have been developed for this purpose. It mainly includes methods for separating plasmid DNA and host DNA by ion exchange chromatography, gel filtration chromatography, fractionation and precipitation. In this laboratory, ion exchange chromatography has been used to obtain extremely high-purity plasmids. In addition, ready-made purified KITs are now available. modern dining table,narrow dining table,dining tables set,dining room furniture Kumusi (Dongguan) Furniture Co., Ltd. , https://www.kcoffeetable.com
Bacterial harvest and purification of lysed plasmid DNA.
1) Large plasmids (greater than 15 kb) are easily damaged, so diffuse lysis should be used to release them from the cells. Bacteria are suspended in sucrose isotonic solution, and then treated with lysozyme and EDTA to destroy the cell wall and outer cell membrane, and then add a detergent such as SDS to dissolve the spherical body. This method minimizes the force required to release the plasmid from inside the bacteria with positive pressure.
2) More vigorous methods can be used to isolate small plasmids. After the addition of EDTA, sometimes the bacteria are exposed to detergent after adding lysozyme, which is cleaved by boiling or alkali treatment. These treatments can destroy the base pairing, so that the host's linear chromosomal DNA can be denatured, but the closed-loop plasmid DNA strands cannot be separated from each other due to the topologically entangled state. When the conditions return to normal, the plasmid DNA strands are quickly and accurately configured to reform into a completely natural supercoiled molecule.
3) Some E. coli strains (such as HB101 variant variants) can release a relatively large amount of sugars when lysed with detergents or heat, when they are subsequently purified by plasmids using cesium chloride-ethidium bromide gradient equilibrium centrifugation Will cause trouble. Sugars will form a dense, fuzzy zone in the gradient next to the position occupied by supercoiled plasmid DNA. Therefore, it is difficult to avoid contamination of the plasmid DNA with sugar, which can inhibit the activity of various restriction enzymes. Therefore, when preparing plasmids in large quantities from E. coli castors such as HB101 and TG1, it is not appropriate to use the boiling method.
4) When preparing plasmids in small quantities from an E. coli strain (endA + strain, such as HB101) expressing endonuclease A, it is recommended not to use the boiling method. Because boiling does not completely inactivate endonuclease A, plasmid DNA will be degraded during subsequent incubation (eg, digestion with restriction enzymes). However, this problem can be avoided by an additional step (extraction with phenol: chloroform).
5) At present, the copy number of this generation of plasmids is very high, so that high yields can be obtained without selective amplification with chloramphenicol. However, some workers continue to use chloramphenicol not to increase the yield of plasmid DNA, but to reduce the volume of bacterial cells in the solution used for mass preparation. A large number of highly viscous concentrated bacterial lysates are troublesome to handle, and adding chloramphenicol to the increase or decrease in the mid-log period can avoid this phenomenon. The amount of plasmid DNA obtained from a smaller amount of cells in the presence of chloramphenicol is approximately equal to the amount of plasmid DNA obtained from a larger amount of cells in the absence of chloramphenicol.