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. 

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... 

No single test is sufficient to detect all possible contaminants and hence multiple procedures must be adopted. Organisms that grow rapidly in cell culture medium are readily apparent when contaminated medium is incubated at 37°C for a few days. Such contamination is not a serious problem as the experiment can quickly be terminated and the contaminated culture eliminated. It is the slow-growing contaminant which produces no obvious change in the medium and which exerts no marked cytotoxic effect, which may be overlooked and yet may dramatically interfere with a biochemical investigation, e.g. satellite DNA bands in CsCl density gradient analyses or unusual forms of enzyme may reflect the presence of a contaminant. 

High-quality algal DNA has been routinely recovered (Fig. 1) in our laboratory using a direct, lysis-fluorochrome dye-CsCl density gradient protocol. This method contrasts with those in which a phenol or isoamyl... 

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. 

The isopycnic method has been used to dramatically demonstrate semiconservative DNA replication, using CsCl density gradients. Separation of DNA, RNA, protein, and carbohydrates can be performed in dense CsCl solutions, where the RNA pellets, the DNA forms bands, and protein and carbohydrates form a thin layer called a pellicle at the top of the gradient. 

When centrifuged to equilibrium in analytical CsCl density gradients, undegraded viral genomes show unimodal, narrow symmetrical bands, as expected for a population of compositionally identical DNA molecules (see Fig. 1.6 for an example). 

Figure 3.1. A. A histogram of the DNA components of the bovine genome. The height of each bar is proportional to the percentage of each component in DNA solid bars correspond to the sharp-melting, open bars to the broad-melting components (see B). The broken line is an enlarged band profile of calf DNA in CsCl density gradient. B. Absorbance-temperature profiles for calf thymus DNA components in 0.1 xSSC (standard saline citrate). Compare the very sharp transitions of 1.705, 1,714 and 1.723 g/cm satellites with the broader ones of 1.697, 1.704 and 1.709 g/cm major DNA components. (From Filipski et al., 1973).

Figure 3.5. Aiialyiical CsCl density gradient profiles of four mouse DNA samples of different molecular weight. Molecular weights were measured from sedimentation velocity experiments except for the highest one which was estimated from the CsCl profile (From Macaya et at., 1976).

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. 

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