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W747599189 abstract "This chapter examines technical considerations associated with the use of 15N in nucleic acid stable isotope probing (SIP) experiments, discusses concerns that should be considered prior to undertaking a 15N-labeling experiment, and provides an overview of different applications of 15N-SIP. The preferable approach is to use quantitative PCR (qPCR) to determine the number of 16S rRNA genes in each gradient fraction. The gene target for qPCR analysis can vary by application, and it may be desirable to use universal 16S rRNA gene-targeted primers or primers that are specific to individual domains, individual subgroups, or genera. In DNA purified by secondary gradient fractionation, nif H genes similar to Methylosinus represented 53% of those recovered while Methylocystis-like sequences represented 17% of those recovered. However, in an experiment in which 15N2-DNA-SIP was used to examine nitrogen-fixing methanotrophs in soil. There are several reasons why 15N-DNA-SIP represents an appealing method for examining nitrogen (N2)-fixing organisms. First, incubations can be carried out at realistic concentrations of substrate, as air can be evacuated from sealed containers and replaced with simulated air containing 15N2. Second, since nitrogen fixation is inhibited in the presence of mineral forms of nitrogen, problems associated with isotope dilution can largely be ignored. Experiments will need to be performed with pure cultures and environmental samples to determine whether 15N-RNA-SIP can be used effectively in microbial ecology studies." @default.
W747599189 created "2016-06-24" @default.
W747599189 creator A5023442989 @default.
W747599189 date "2014-04-30" @default.
W747599189 modified "2023-09-26" @default.
W747599189 title "Stable Isotope Probing Techniques Using 15N" @default.
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W747599189 doi "https://doi.org/10.1128/9781555816896.ch7" @default.
W747599189 hasPublicationYear "2014" @default.
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