[DNA] ultracentrifugation

[DNA] ultracentrifugation

Modern plasmid DNA isolation and purification by separation from the E. coli represented, 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 DNA a quality control In recent years in the ultra-centrifuge technology is an important topic. Fast and plasmid DNA isolation and purification equipment and the ultra-centrifugation (speed centrifuges, the old and ancillary equipment) has put a higher demand.

E.coli is a typical prokaryotic cell biology, because of the lack of prokaryotic cells with its nuclear cells from the kind of film units composed of a variety of functional components can be separated for the specificity of regional and local independent of the endometriosis, therefore not covered by its nuclear cell of the cell (nucleus, endoplasmic reticulum, golgi apparatus, the line of Rafah, lysosomes, etc.). SEM micrographs showed that E.coli can be two different regions within the cytoplasm and the nucleus and cytoplasm January 1, in their thin layer around the outside of the cell membrane and very thick cell wall and cell wall in the end some external attachment of the free flagella. Plasmid DNA in the nucleus to filamentous exist, such filaments bar in a variety of circumstances is very long circular DNA fragments some of the folding up of poly body.

Microstructure against E.coli question, in ultra-centrifugal separation and purification of plasmid DNA sequence pretreatment before: E.coli → → with lysozyme to the cell wall using surfactants such as SDS, Trit X-100 membrane → EE, such as acetic acid used pot to DNA, RNA and protein precipitation majority (more than 90%).

Sediments can be joined in TE buffer (10-m-MTris HCL lmMEDTA, pH8.0) to live on protein molecular sieve to RNA; also can be used to ultracentrifugation protein Habitat to RNA to DNA-level DNA fragments or .

[Ultracentrifugation Plasmid DNA Isolation Method]

The traditional separation methods: a few years ago, due to equipment constraints, plasmid DNA from the general balance by CsCl density centrifugation, since the formation of gradient. 10 ~ 12 ml single-tube capacity as an example, using the old left-separation, 36.000 rpm × 60-hour separation with the old angle-45, OOOrpm × 36 hours, including acceleration and deceleration, the former spent 130 million to the Department of Driver Life, which spent 100 million to the drive to life, and this life was speeding centrifuge total of 100 ~ to 20 billion, no doubt each with excessive costs, coupled with CsCl consumption, high cost and other factors so that such separation and purification work a very expensive experiment.

[Plasmid DNA ultracentrifugation from the latest developments:

(1) speed of the old vertical centrifugal separation (titanium alloy or carbon fibre): From 1975 to the old vertical tube WHO, in recent years the major development of the centrifuge manufacturer of the old vertical, single-tube capacity 0.2 Oml to 4 ml, the maximum speed from 50,000 rpm to 120,000 rpm, RCFmax up to 700, OOOXg 1990s and the development of new models has been able to make the old vertical tube plasmid DNA test done centrifugal separation easier.

(2) near-vertical centrifugal separation of the old: In order to remove the old vertical tube used for plasmid DNA in the wall of the centrifuge formation of RNA precipitation has been formed on the DNA zone pollution, but also to improve the old-general diagonal (dip 25 • - 35 •) from the settlement because of a longer and therefore a longer time separated the shortcomings of the past few years the development of a wide range of near-vertical pipe the old (Near VerticalTube Rot, as the old NVT or Neo Angle Rotor, small angle at the old, or NT). their profile centrifuge tube and centrifuge central axis drive in the angle between the axis of 7.5 • - 10 • between speed from 120 to 65,000 rpm, OOOrpm, RCFmax up to 646000 × g single-tube capacity from 2 ml to 13.5 ml. NVT (or NT) is the development of the old plasmid DNA isolation and for the design, of course, also applies to mitochondrial DNA, chromosomal DNA, RNA and serum lipoprotein • Isolation and purification.

(3) discontinuous gradient separation ladder: DNA Isolation and Purification of the school is the traditional method used since the formation of CsCl density gradient centrifugation balance, at the beginning of the centrifuge tube CsCl density uniform, uniform distribution of these samples.