Isolation and partial characterization of two cryptic plasmids from an extreme thermophile
Isolation and partial characterization of two cryptic plasmids from an extreme thermophile
复制标题
从极端嗜热菌中分离和部分表征两个隐秘质粒
DOI：
10.1042/bst0130214
复制
发表时间：
1985
影响因子：
3.9
通讯作者：
Ralph A. D. Williams
中科院分区：
文献类型：
--
作者：
N. Raven;Ralph A. D. Williams
Two plasmids have been isolated from an extreme thermophile, ascribed to the genus Thermus and designated YS045 by Sharp et al.(1983). The following procedure was developed. Shake-flask cultures (500ml) of Thermus YS045 in Ramaley & Hixson medium (1970) were agitated at 150rev./min at 65 C in a rotary incubator. Harvested cells, for each litre of culture grown, were resuspended in 20ml of 25% sucrose solution and, after the addition of 2ml of a lOmg of lysozyme/ml solution, held at 42 C for 20min. Six millilitres of 0.25~-disodium EDTA, pH 7.0, was then added. Lysis was brought about by the addition at room temperature of 3ml of a 20% sodium dodecyl sulphate solution. Sodium hydroxide (5M) was then added dropwise to pH 12.1-12.3. After 5min 20ml of 3 M-sodium acetate solution (pH 6.0) was used to restore neutrality. The resultant suspension was held at 4 C for 30min before centrifugation at 20000g for l0min. Ethanol (2vol.) was added to the supernatant which was then incubated at-20 C for 1 h before centrifugation at lOOOOg for 5min. The pellet obtained was subjected to a second alkaline denaturation (Marko et al., 1982). The ethanol precipitable material remaining was dissolved in TE buffer (50mM-Tris, 1 mM-EDTA, pH 8.0) and an equal volume of 5M-ammOniUm acetate added. After 20min at 4 C the mixture was centrifuged at 20000g for l0min. The supernatant was then treated with ethanol as above and the precipitate taken up in TE buffer. The material obtained, consisting almost entirely of low-M, RNA and plasmid DNA, was then run on a 0.8% agarose gel in 0.04 M-Tris acetate buffer, pH8. 3. After staining with OSpg of ethidium bromide/ml and under UV illumination, gel slices containing plasmid DNA were cut out and the material electroeluted from them within dialysis bags. The DNA solutions obtained were then subjected to caesium chloride/ethidium bromide equilibrium density centrifugation (Maniatis et al., 1982) to separate the DNAs from soluble impurities, mainly acidic polysaccharides, co-eluted from the gel slices.
