CsCl/ethidium bromide

Visualize the DNA band with a long-wavelength UV lamp. Remove the DNA to 5-ml heat seal tubes, fill the tubes with CsCl-ethidium bromide solution (1.55 g/ml) and reband at 58,000 rpm for 6 hr or 44,000 rpm for 12 hr at 20° in a VTi 65 rotor (Beckman). 

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]. 

MtDNAs are purified by CsCl-ethidium bromide ultracentrifugation at 85,000 g for 36 hr at 20°C. Fractions containing closed circular mtDNA molecules are collected, extracted four times with H20-saturated butanol to remove the dye, diluted with an equal vol of H2O and precipitated with 2 vol of ethanol at -20°C. 

Standard protocols for rapid extraction of recombinant plasmid DNA from bacterial cultures (6), omitting further purification by equilibrium centrifugation in CsCl-ethidium bromide gradients, give DNA substrates of sufficient purity (see Chapter 9). 

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. 

Add 15.4 g CsCl and 0.4 ml of 5 mg/ml ethidium bromide. Centrifuge for 40 h at 150,000 g. Remove the prominent lower band (clearly visible under long-wave UV illumination) preferably... 

The DNA can he seen directly in the CsCl gradient by reversibly staining with ethidium bromide (Firtel and Bonner 1971). Solid CsCl is added to the DNA solution to make the density up to about 1.55 g ml (0.97 g CsCl to each 1 ml original DNA solution) followed hy 1/20 volume of ethidium bromide solution (10 mg/ml in water). Spinning time is 48 hr at 40,000 rev/min. RNA and... 

Classical Method. This method (Rl) involves isopycnic centrifugation of cleared lysate in a solution of CsCl containing ethidium bromide (EtBr). EtBr binds by intercalating between DNA base pairs, which causes the DNA to unwind. A covalently closed circular (ccc) DNA molecule such as a plasmid has no free ends and can only unwind to a limited extent, thus limiting the amount of bound EtBr. Linear DNA, such as fragmented chromosomal DNA, has no such topological constraints and can therefore bind more of the EtBr molecules. Because the density of the DNA/EtBr complex decreases as more EtBr is bound, and because more EtBr can be bound to a linear molecule than a covalent circle, the... 

Cesium chloride CsCl and/or ethidium bromide contamination time consuming not readily scaled up (80,161)... 

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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