Density Gradient Sedimentation Equilibrium

The distribution of N and N was revealed by the technique of density-gradient equilibrium sedimentation. A small amount of DNA was dissolved in a concentrated solution of cesium chloride having a density close to that of the DNA (1.7 g This solution was centrifuged until it was nearly... 

The DNA in a bacterium is uniformly labeled with N, and the organism shifted to a growth medium containing N-labeled DNA precursors. After two generations of growth, the DNA is isolated and is subjected to density-gradient equilibrium sedimentation. What proportion of light-density DNA to intermediate-density DNA would you expect to find ... 

During early studies of the denaturation of double-strand DNA, it was not knovra whether the two strands unwind and completely separate from each other. Suppose that you have double-strand DNA in which one strand is labeled vHth N and the other is labeled "with N. If density-gradient equilibrium sedimentation can be used to distinguish between both double- and single-strand molecules of different densities, how can you determine whether DNA strands separate completely after denaturation ... 

Although the system described could yield P-labeled daughter DNA molecules, chemical methods cannot distinguish DNA in which both strands are radioactively labeled from DNA in which one strand is labeled and one strand is unlabeled. In their experiments, Meselson and Stahl used a physical technique, density gradient equilibrium sedimentation, to separate the labeled molecules according to their content of N and N, which differ in their specific densities. 

Equilibrium sedimentation technique working with a multi-component solvent forming a density gradient in a centrifugal field... 

Particles in the gradient may be separated on the basis of sedimentation rate a sample introduced at the top of the preformed gradient setdes according to density and size of particles, but the run is terminated before the heaviest particles reach the bottom of the tube. If the density of all the particles lies within the range of the density limits of the gradient, and the run is not terminated until all particles have reached an equilibrium position in the density field, equilibrium separation takes place. The steepness of the gradient can be varied to match the breadth of particle densities in the sample. 

MWD can be expressed by several equivalent formulas derived from the theory of equilibrium sedimentation at ideal conditions. In the following the well-known Fujita formalism (see Equation 2) will be used. This expression related the density of MWD, f(M), to concentrations or concentration gradients, respectively ... 

This standard method of density gradient centrifugation, however, has a serious disadvantage. Because of the sedimentation and diffusion equilibrium formation of the density profile the time needed for orie experiment amounts to more than 15 hours. 

M. Meselson, F. W. Stahl, and J. Vinograd, Proc. Natl. Acad. Sci. US, 43 581 (1957). Equilibrium Sedimentation of Macromolecules in Density Gradients. 

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