Cesium chloride concentration gradient

Fig. 2.4 Flowchart of the steps for rAAV production as described in Protocol 1. HEK293 cells are expanded, transfected, and harvested at 60h posttransfection. The cells are lysed, and loaded onto either a cesium chloride gradient or an iodixanol gradient to separate infectious virions from empty capsids. Virus is purified using either heparin affinity chromatography or ion-exchange chromatography. Virus preparations are formulated and concentrated, and characterized...

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 targeting vector needs to be linearized for electroporation. The DNA is prepared by standard maxiprep procedures, for example, cesium chloride gradient centrifugation, or popular kits (such as Qiaex, Promega Wizard, Geneclean). Ethanol-precipitate the digested DNA before the electroporation, and dissolve the pellet in sterile TE. The concentration of the DNA should be around 1 J.g/liL. 

The density of one molecule of DNA rich in guanine-cytosine pairs is greater than that of a DNA rich in adenine-thymine pairs. Thus, if a DNA suspension composed of a mixture of two different DNA s—one rich in AT pairs and another rich in CG pairs—is centrifuged in gradient concentrations of cesium chloride, the two types of DNA band separately at the end of the centrifugation. The position of the band depends on the bouyant density of the DNA molecule. The bouyant density of the DNA molecule was shown to be directly proportional to the amount of CG pairs present. The determination of the bouyant density of DNA has thus provided a convenient method for establishing quickly the base composition of small amounts of DNA. 

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