Centrifugation CsCl density gradient

Ion-exchange HPLC can also be useful in the separation of larger nucleic acid molecules. One such application is as an alternative to CsCl density gradient centrifugation in the preparation of plasmids. Plasmid molecules typically consist of between 1000 and 10 000 base pairs. The plasmid is first isolated from the bacterial cell by alkaline lysis and pure plasmid obtained from this crude extract by a one-step chromatographic separation. 

Fig. 6.1. (a) CsCl density gradient centrifugation separates DNA from RNA and proteins due to their different buoyant densities, (b) By addition of ethidium bromide, chromosomal DNA can be separated from plasmid DNA. 

The differences in the buoyant density of biomacromolecules can be exploited for their separation/purification and characterization. The density of DNA is slightly greater than 1.7g/mL, while the density of RNA is more than 1.8g/mL. Proteins have densities less than 1.3g/mL. Therefore, DNA, RNA and proteins can be separated by the CsCl density gradient centrifugation (-200000 x g), also known as isopycnic centrifugation. Since single-stranded DNA (ssDNA) is denser than double-stranded DNA dsDNA), the isopycnic centrifugation can be used to separate the randomly coiled ssDNA and helical dsDNA, which also forms a narrower band. The buoyant density (p) of a DNA molecule, which is related to its G -i- C content by... 

Bemardi et al. (1985) have made extensive studies of the genomes of a wide range of eukaryotes using this method. Before carrying out the CsCl density gradient centrifugation they reduced the average... 

The buoyant density of informosomes from sea urchins was found to be higher, 1.50 to 1.75 g/cm, which corresponds to a much lower protein content (Infante and Nemer, 1968). It may be the result of the higher salt content in sea urchin cells (0.5 A/KCl, for example, may produce a partial dissociation of proteins from the 308 particles). Another explanation is the possible removal of some proteins from informosomes during CsCl density gradient centrifugation in the absence of formaldehyde (8pirin, 1969). 

To reduce the percentage of wild-type DNA, it may be necessary to use the super-coiled version of the plasmid, purified either by CsCl-density gradient centrifugation or from agarose gels by the GeneClean protocol... 

DNA extracted and centrifuged to equilibrium in CsCl density gradient... 

Some 37 samples from 16 coals of the Pennsylvania State University coal data base (PSOC) were examined. Separate density fractions were obtained by isopycnic density gradient centrifugation of small ( 3iim) coal particles in an aqueous CsCl density gradient. (4J The individual samples are listed by PSOC numbers, coal description, ASTM designation of coal-rank maceral type, and density in Table I. After separation, the samples... 

Of considerable further interest is the finding of Marmur and Ts o (1961) that the denaturation of DNA in formamide-water mixtures results in the separation of the two polynucleotide strands. An N N hybrid Escherichia coli DNA was treated with 95% formamide at 37°C for 15 min at an ionic strength of 0.01, the formamide was then removed by dialysis, and the DNA was centrifuged in a CsCl density gradient. The presence of two bands of density different from that of the original hybrid indicates that strand separation occurred. It is of considerable practical interest that these conditions are much milder than those required to achieve strand separation in aqueous solution. Whether strand separation occurs also in solutions of DNA in other nonaqueous solvents is not yet determined. 

Native apoferritin can be prepared by a number of centrifugal methods. Density gradient centrifugation (62) in isopycnic gradients of CsCl or in sucrose have been used to fractionate native ferritin according to its iron content. The native apoferritin sediments with a buoyant density of 1.23, whilst full ferritin has a density in excess of 1.8. Typically using an SW 27 rotor 100 hr are required at 25.000 rpm with a CsCl... 

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