CsCl gradients

Satellite DNA. A DNA fraction the base composition of which differs from that of the main component of DNA, as revealed by the fact that it bands at a different density in a CsCl gradient. Usually repetitive DNA or organelle DNA. 

Physcomitrella patens (moss) Cultures gametophyte tissue sucrose and CsCl gradients Pure cpDNA other methods failed 26... 

Alternatively, secondary gradient purification can be performed immediately after Step 6 or 7 by loading the small amount collected by either procedure onto a 1.56 g/ml CsCl gradient. The gradient is then spun again at 36,000 rpm for 42 hr. 

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. 

Also, as a word of caution, it should be noted that nucleic acids precipitated by PEG can contain macromolecular impurities originating from the PEG these impurities are not detectable by the usual methods (e.g., electrophoresis or UV absorption spectroscopy) and they cannot be readily removed even by CsCl gradient centrifugation. 

Figure 9-15. Poly I,G-effected separation of the complementary strands of A phage DNA in preparative and analytical CsCl gradients. Upper trace represents the absorbance (260 nm) of the 4-drop (50 tl) fractions (total volume 2.5 ml) measured in a 20 /u.1 microcuvette (2 mm light path). Lower trace represents the microdensitometric tracing of the photograph of the same undiluted material banded in the analytical ultracentrifuge (4°C, 3 mm cell) with added density marker DNA (Cytophaga johnsonii, 1.6945 g/cm dashed line). Peak C contains the DNA strands C, which preferentially bind poly I, G the complementary strands W band under peak W. Symbols dN and NN indicate the positions (densities) of the denatured and native Acbj DNA, respectively. [From Z. Hradecna and W. Szybalski, Virology, 32 633 (1967).]...

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... 

Fig. 11.4. Ultraviolet absorbance measurements on individual fractions from a CsCl gradient. The centre peak is DNA. The absorbance at the bottom of the gradient is probably due to RNA.

Figure 2. Predicted buoyant position of hydrated constituents in CsCl gradients.

Figure 4. Preparative 36% and 39% CsCl gradients of Brucella LPS. Shows separation of other components. Left 39% gradient showing B. meletensis LPS. Right 36% gradient showing B. abortus LPS.

Figure 5. 34% CsCl gradients of Brucella LPS. The LPS from B. abortus strain indicated under tube.

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