ISOLATION OF COVALENTLY CLOSED CIRCULAR DNA OF HIGH MOLECULAR-WEIGHT FROM BACTERIA
ISOLATION OF COVALENTLY CLOSED CIRCULAR DNA OF HIGH MOLECULAR-WEIGHT FROM BACTERIA
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DOI：
10.1016/0003-2697(76)90338-9
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发表时间：
1976-01-01
影响因子：
2.9
通讯作者：
NESTER, EW
中科院分区：
文献类型：
--
作者：
CURRIER, TC;NESTER, EW
本文详细介绍了一种从农杆菌中高效提取高相对分子质量共价闭合环DNA(CCC-DNA)的简便方法。虽然这种方法是为从农杆菌中分离MW>；108道尔顿的CCC-DNA而开发的，但该技术也适用于从各种其他细菌中分离不同大小的CCC-DNA。该技术包括对染色体DNA进行剪切和碱变性，然后通过苯酚提取优先去除单链DNA。剩下的DNA主要是ccc-DNA。然后浓缩DNA，在氯化铯-溴化乙锭密度梯度中通过离心法将CCC-DNA从染色体DNA中分离出来。利用该技术，还从大肠杆菌、根瘤菌、柠檬酸杆菌和干酪乳杆菌中提取了不同大小的CCC-DNA。
A simple method is described in detail for the efficient isolation of high molecular weight covalently closed circular DNA (ccc-DNA) from Agrobacterium [tumefaciens]. Although this method was developed for isolating ccc-DNA of MW > 108 daltons in Agrobacterium, the technique is also useful in isolating ccc-DNA of varying sizes from a variety of other bacteria. The technique involves the shearing and alkali denaturation of the chromosomal DNA, followed by the preferential removal of the single-stranded DNA by phenol extraction. The DNA which remains is largely ccc-DNA. The DNA is then concentrated, and the ccc-DNA is separated from the chromosomal DNA by centrifugation in a CsCl-ethidium bromide density gradient. By this technique, ccc-DNA of varying sizes was also isolated from Escherichia [coli], Rhizobium spp., Citrobacter sp., and Lactobacillus [casei].