Ethidium bromide centrifugation

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... 

Isolation of Plasmid Enriched DNA. The three strains of B. megaterium were cultured in yeast-soy broth (4% Bacto-yeast extract and 1.6% Bacto-soytone from Difco Laboratories, Detroit, pH 7.2). Plasmid DNA from each strain was isolated and purified by cesium chloride-ethidium bromide equilibrium density centrifugation following the procedure of Carlton and Brown ( ). Subsequent gel electrophoresis (0.5% agarose) revealed the presence of some chromosomal DNA contamination. 

Nucleic acids can be visualized by ethidium bromide staining, UV shadowing, or phosphorimaging of radioactive samples. A sterile scalpel should be used to excise the separated product which can then be eluted by electrophoresis (1 x Tris-borate, pH 8.3) into a 30kDa molecular weight cutoff (MWCO) filter (Millipore). The product is concentrated by centrifugation, dialyzed, and resuspended in the buffer of choice. The yield of nucleic acid is typically 75 %, and ethanol precipitation is not needed. 

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... 

Plasmid purification Ultra centrifugation (mutagenic reagents and ethidium bromide) lEC (gravity flow columns provided in commercial kits) RPC (organic, toxic solvents) tEC and/or SEC (use only GRAS reagents)... 

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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