期刊
NATURE PROTOCOLS
卷 2, 期 4, 页码 860-866出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2007.109
关键词
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资金
- Natural Environment Research Council [NE/C507902/1, NER/A/S/2002/00876] Funding Source: researchfish
Stable-isotope probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-isotope-labeled carbon (C-13) or nitrogen (N-15) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids ( DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4-5 days, with much of the time being committed to the ultracentrifugation step.
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