Preparative CsCl gradient isolation

To isolate genomic DNA from E. coli, the cells are treated with lysozyme and then lysed by SDS in the presence of proteinase K. Proteinase K, which is active even in SDS solution, degrades proteins including nudeases. Cell debris, polysaccharides and unhydrolysed protein are removed by precipitation at room temperature with cetyltrimethylammonium bromide (CTAB). DNA is isolated from the supernatant by precipitation with alcohol. RNA can be removed from DNA preparations by incubation with DNase-free RNase. Further purification can be effected by a phenol/ chloroform/isoamyl alcohol (25 24 1) extraction, and/or by CsCl gradient centrifugation (see Sect. 4.3.4.2 ) to remove the remaining protein and RNA. 

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. 

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