A method for the preparation of DNA from calf thymus nuclei in maximum purity and yield, analysis of this DNA compared with that obtained by other methods.
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%.
Welsh
R S
RS
Institut für Medizin, Forschungszentrum, Jülich, Germany.
D000818:Animals; D002417:Cattle; D002467:Cell Nucleus / Q000737:chemistry*; D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D006868:Hydrolysis; D007202:Indicators and Reagents; D009687:Nuclear Proteins / Q000302:isolation & purification; D010636:Phenols; D011402:Pronase; D012260:Ribonucleases; D012965:Sodium Chloride; D013056:Spectrophotometry, Ultraviolet; D013950:Thymus Gland / Q000737:chemistry* / Q000648:ultrastructure
D007202:Indicators and Reagents; D009687:Nuclear Proteins; D010636:Phenols; D012965:Sodium Chloride; D004247:DNA; D012260:Ribonucleases; D011402:Pronase