DNA
微核试验
色谱法
DNA提取
分类
单元格排序
超离心机
化学
电泳
蔗糖
分子生物学
分离(微生物学)
细胞
生物化学
生物
聚合酶链反应
基因
生物信息学
计算机科学
有机化学
程序设计语言
毒性
作者
Michael Abend,S. Frombeck,Claudia Baaske,D. van Beuningen
出处
期刊:Mutation research
[Elsevier]
日期:1996-05-01
卷期号:360 (1): 23-28
被引量:3
标识
DOI:10.1016/s0165-1161(96)90233-2
摘要
Micronuclei (MNC) of L929 cells were isolated 72 h after irradiation with 6 Gy for characterization of their DNA content, using gel electrophoresis. A novel method for isolation of MNC based on a sucrose gradient ultracentrifugation was developed. With this efficient method (80% recovery) more than 150 x 10(6) MNC per day could be isolated. The purity was > 99%. However, the low number of main nuclei in the MNC isolate ( < 1%) resulted in a contamination of MNC DNA with about 15% main nucleus DNA, due to the several times higher DNA content of main nuclei. Cell sorting was utilized to maximize the purity by choosing the recommended sorting mode for highest purity. Isolation of MNC with the cell sorter was successful (100% purity), but also time-consuming (1-2 x 10(6) MNC per working day could be isolated) and insufficient (10% recovery). Extraction of DNA of these isolated MNC resulted in less than 1 ng/day. Hence, at least 1 week of cell sorting would be necessary for one electrophoretic run. When employing the sucrose gradient method, 2000 times more DNA of MNC have been isolated. We therefore consider this method as the most efficient way for rapid and low cost isolation of large amounts of purified ( > 99%) MNC without the employment of sophisticated and expensive techniques (cell sorting) and the accompanied knowhow. In contrast, maximum purity but low yields of MNC can be obtained by cell sorting.
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