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By Walker J.M. (ed.)
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The most usual way of checking the success of such procedures is by looking at the products using electrophoresis in agarose gels. This process separates DNA molecules by size, and the molecules are made visible using the fluorescent dye ethidmm bromide. In this way DNA can be checked for size, intactness, homogeneity, and purity. The method IS rapid and simple, yet capable of high resolution, and IS so sensitive that usually little of the sample 1s needed for analysis Agarose forms gels by hydrogen bonding when m cool aqueous solution, and the gel pore size depends on agarose concentration When DNA molecules are moved through such a gel by a steady electric force, their speed of movement depends almost entirely on their size, the 43 44 Boffey smallest molecules having the highest mobrlmes.
Introduce gas slowly so that it condenses. Store the tube with frozen Freon-12 on liquid nitrogen. 2. The preparatron of concentrated cells should be carried out in a cold room at +4”C. Suspensron cultures are chilled by pouring the warm culture onto one-half the culture volume of frozen PBS. The chilled cells are washed twice m cold PBS by centrifugation (2OOOg, 2 mm, 4°C). The final pellet of cells is resuspended by adding one pellet volume of PBS or water (Note 2). The cells are chilled, washed, and frozen as rapidly as possible (see step 4 below) 3.
P. Mathew MRC Molecular and Cellular Cardiology Research Unit, University of Stellenbosch Medical School, Tygerberg, South Africa Introduction The isolation of high molecular weight eukaryotic DNA m good yield is an Important prerequisite for the analysis of specific sequences by Southern blotting (Chapter 9), or for molecular cloning m phage or cosmld vectors (Chapter 49). In the procedure described below, cells from the organism are disrupted by homogemzatlon m Trlton X-100 and the nuclei pelleted by centrifugation The nuclei are then resuspended and treated with SDS, which dissociates the DNA-protem complex.
Nucleic Acids by Walker J.M. (ed.)
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