DNA school

DNA ultracentrifugation] [Edit this paragraph] Modern separation and purification of plasmid DNA from E. coli to isolated as the representative, in view of the overall situation bacilli (E.coli) in the molecular biology of the important position in the separation and purification from E.coli quality control in recent years become a DNA Ultra-centrifuge technology in a very important subject. The plasmid DNA and the rapid separation and purification of the ultra-centrifuge equipment (centrifuges speeding, Zhuantou and ancillary equipment) has put forward higher requirements. E.coli is typical of the original cells, the cells because of the lack of its nuclear cells are the kind of film by units composed of a variety of functions can be separated for specific components of the regional and local independent endomembrane system, Therefore not covered by its nuclear cell of the cell (nuclear, ER, Gorky body, the line of Rafah, the lysosome, etc.). SEM micrographs showed that E.coli can distinguish between the two regions within the cytoplasm and nuclear transfer on January 1, in their thin layer around the outside of the cell membrane and very thick walls, some attached to one end of the external walls of the free Flagellum. Plasmid DNA in the nuclear area, to fine filaments exist, such filaments bar in a variety of circumstances is extremely long circular DNA fragments by some of the folded-up of body. E.coli for the microstructure of question, in ultra-centrifuge separation and purification of plasmid DNA sequence before the pretreatment is: E.coli → lysozyme to use the cell wall → with surfactant such as SDS, Trit X-100 and other EE membrane → pot with acid so that DNA, RNA and protein precipitation majority (90 percent). Sediments can join TE buffer (10 m-MTris-HCL lmMEDTA, pH8.0) to live on after the zeolite protein to RNA; can also be used to ultracentrifugation of Habitat, to RNA, DNA-to-class or DNA fragment . [Plasmid DNA ultracentrifugation the separation method: The traditional separation method: A few years ago, due to equipment constraints, the plasmid DNA from the general balance, with CsCl density centrifugation, since the formation of gradient. To 10 ~ 12 ml single-tube capacity as an example, with Lut-Zhuantou separation, 36.000 rpm × 60 hours, with angle-Zhuantou Separation 45, OOOrpm × 36 hours, including a slowdown, the former sharing to 130 million drives to the Department of Life , Who also spent 100 million drive to the Ministry of life, which was speeding on the life of 100 centrifuges to transfer 20 billion, no doubt each test costs too high, with CsCl amount, your prices and other factors, That such separation and purification of a very expensive experiment. [Plasmid DNA speeding centrifuge - the latest progress (1) speeding vertical tube Zhuantou the centrifuge (Chin alloy or carbon fiber manufacturing): From 1975 to the vertical pipe Zhuantou WHO, the main centrifuge production in recent years to develop the vertical pipe Zhuantou, single-tube capacity of 0.2 ml to 4 Oml, the maximum speed from 50000 rpm to 120000 rpm, RCFmax up to 700, OOOXg, 90 in the development of new models and Zhuantou plasmid DNA has been able to make vertical centrifuge tube experiments done handy. (3) near vertical pipe Zhuantou centrifuge: In order to eliminate the vertical centrifugal Zhuantou for plasmid DNA in the wall of the formation of the RNA has precipitated the formation of DNA zone of pollution, but also to improve the general oblique angle-Zhuantou (Dip 25 • - 35 •) from the settlement because of a longer and therefore have a longer time from the shortcomings of the past few years developed a variety of near-vertical pipe Zhuantou (Near VerticalTube Rot, referred to NVT Zhuantou or Neo Angle Rotor, small angle Zhuantou false, or NT). Centrifugal their central axis and the vertical section of the centrifuge drive in the angle between the axis between 7.5 • - 10 • between speed from 65000 rpm to 120, OOOrpm, RCFmax can 646000 × g of single-tube capacity from 2 ml to 13.5 ml. NVT (or NT) Zhuantou primarily for the development of plasmid DNA separation design, of course, it also applies to mitochondrial DNA, chromosome DNA, RNA and serum lipoprotein • separation and purification. (3) non-consecutive ladder gradient separation: separation and purification of DNA school is using the traditional method since the formation of CsCl gradient balance, density centrifugation, centrifugal at the beginning of the density of CsCl homogeneous, uniform distribution of these samples.