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In the preparation of calf thymus DNA, a method (1) was developed to yield DNA in maximum yield and purity, with a minimum of shear degradation, which differed in three respects from those used in standard preparations: 1) use of calf thymus nuclei rather than whole tissue as a source of the DNA; 2) use of initial Pronase, then on the next cycle RNAase + Pronase digestions; 3) use of optimal concentrations of reagents to precipitate denatured proteins in the purification cycles. The optimal reagent concentrations were determined in tests by changing each variable one at a time to be 3M NaCl, 3% sarcosyl. The purification cycles consisted of adjustment of DNA solution in buffer to 3M NaCl-3% sarcosyl, centrifugation, alcohol precipitation, and redissolving DNA fibers in buffer. The DNA from nuclei, digested with Pronase then with RNAase + Pronase was then subjected to five additional purification cycles. The purified DNA thus obtained (N-DNA) had a protein content of 0.64%. When purified calf thymus nuclei are subjected directly to salt-sarcosyl purification cycles, the initial protein content is about 21%, but after the second cycle the percentages of protein fell off in subsequent cycles in a parallel manner as compared to the DNA obtained by the Blin and Stafford method (2) and then subjected to NaCl-sarcosyl purification. The degree of purity obtained by these purification methods was appreciably greater than obtained for a modified method of Gross-Bellard et al. (3). Two portions of DNA purified by this method were analyzed: 1) dialyzed 11 times after the Proteinase K digestion, and 2) dialyzed 6 times. Portion 1) gave a protein content of 0.96% and a yield of 0.135% (g/100 g tissue). Portion 2) gave a protein content of 1.34% and a yield of 0.133%. |
