フェノール法による家蚕組織核酸の調製

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It has been difficult to prepare nucleic acids from silkworm tissues. Especially, preparation of DNA in fibrous state was unsuccessful so far. By modified phenol procedure of Kirby, nucleic acids were prepared pretty easily from silkworm tissues. Tissues were homogenized in Ca-free Tyrode's solution with a Waring blender and centrifuged. The extract was mixed with the same volume of 90 % phenol, stirred for an hour at room temperature and centrifuged. Sodium acetate was dissolved in the aqueous extract, upper layer, to make a final concentration of 2 % and 2 volumes of ethyl alcohol were added to precipitate RNA. The precipitate was dissolved in 10 % CaCl_2 and centrifuged for 30 min. at 10,000 r.p.m. The clear supernatant was mixed with 0.3 volume of alcohol and centrifuged. Into the supernatant, alcohol was further added to make total volume of added alcohol 2 times of CaCl_2 solution. The precipitate was gathered, dehydrated by the repeated treatment with alcohol of the increasing concentration step by step, washed with ether and dried in vacuo. Thus, RNA powder of about 50 to 60 % of purity was obtained. DNA was also prepared by modified Kirby's method. Silkworm tissues, fresh or frozen, were homogenized in 6 % p-aminosalicylic acid, centrifuged and the supernatant was treated with 90 % phenol as above. After centrifuging, PAS-layer was mixed with ethoxyethanol and nucleic acids fraction was precipitated. The fraction was dissolved in conc. NaC1 solution, deproteinized and nucleic acids were reprecipitated. Similarly, the precipitate was dissolved in 10 % CaCl_2 and insoluble matter was discarded. Into the supernatant, 0.3 volume of alcohol was added and fibrous DNA precipitate was obtained. DNA was reprecipitated from NaCl solution with alcohol and dried in vacuo after dehydration with alcohol. It was more effective that DNA was prepared from cellnuclei which had been preliminarily isolated from worm tissues by citric acid method than from whole tissues. Thus, RNA and DNA were prepared by these combined procedure of phenol and CaCl_2 treatments, principally two steps. CaCl_2-alcohol treatment was found to be effective in rough separation between RNA and DNA. Moreovere this treatment is seemed to be necessary to prepare DNA preparation of fibrous state. These preparations of nucleic acids could be further purified by the usual method.Kirby氏のフェノール法によつて家蚕組織から核酸を甚だ簡単に調製することが出来た。特に従来調製に成功していなかつたDNAの糸状標品がフェノール処理にCaCl_2処理を組合わせることによつて調製された.更にこの方法は組織全体についても有効であるが細胞核を予め分離しこれから調製すればすぐれた標品が得られた.
九州大学の論文

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大村浩久
九州大学農学部

フェノール法による家蚕組織核酸の調製

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