Plasmids ultracentrifugation

Deoxyribonucleic acid (from plasmids). Purified by two buoyant density ultracentrifugations using ethidium bromide-CsCl. The ethidium bromide was extracted with Et20 and the DNA was dialysed against buffered EDTA and lyophilised. [Marmur and Doty J Mol Biol 5 109 1962 Guerry et al. J Bacteriol II6 1064 1973.] See p. 504. 

Toyopearl HW-75 resin, with pores larger than 1000 A, have been used in place of ultracentrifugation steps for the purification of plasmid DNA. Ultracentrifugation is a time-consuming process and requires expensive chemicals, such as cesium chloride. Toyopearl HW-75 resin provides superior separation performance for plasmid DNA and also provides high yields (54). 

The supercoiled fraction obtained by CsCl-ethidium bromide equilibrium ultracentrifugation of Halobacterium halobium DNA contains, in addition to the characterized plasmids, a heterogeneous population of minor ccc DNAs . It is supposed that these arise by intramolecular recombination of the chromosome and it is not known whether they are replicated [26,27]. 

Once the bacterial cells have been lysed and a crude preparation of plasmid has been obtained, several other methods are available for purification of plasmid from the crude nucleic acid preparation, such as cesium chloride ultracentrifugation, gel filtration/size exclusion chromatography, anion exchange (used in the Qiagen kit mentioned above), and HPLC (Table 2) (see Note 2). 

Ultracentrifugation (isopycnic) DNA strand separation, first pure gene preparation of plasmid/viral DNA... 

Deoxyribonucleic acid (from plasmids). These are purified by two buoyant density ultracentrifugations... 

Other methods tend to extract total DNA (or DNA only enriched in mtDNA) and to separate mtDNA and nuclear DNA on the basis of their differential properties. One way for the preparation of the mtDNAs is an adaptation of the method of separation of plasmid DNA from the bacterial chromosome by denaturation / renaturation. An alkali is used to denature the DNA (Bimboim and Doly 1979). Then, the mixture is neutralized the mtDNA reanneals rapidly whereas nuclear (linear) DNA partially renatures in a complex network and forms, with proteins and SDS, an aggregate which can be removed by centrifugation. Another way involves an ultracentrifugation, as indicated above, in CsCl with intercaling dye. As the concentration of mtDNA relative to nuclear DNA is usually low in somatic cells (1% of the total DNA), it is useful either to enrich the initial preparation in mtDNA and to repeat the step(s) of purification. 

Total cellular DNA from B. stearothermophilus and plasmid DNA were isolated by previously described methods [11,12] and purified by CsCl gradient ultracentrifugation or by Sephacryl S-1000 chromatography [13]. Phages and phage DNAs were isolated and purified by standard methods [10,11]. 

---