Abstract：
:DNA-SIP (DNA-based stable isotope probing) is increasingly being employed in soil microbial ecology to identify those microbes assimilating the 13C/15N labelled substrate. Isopycnic gradient centrifugation is the primary experimental process for conducting DNA-SIP. However, diverse centrifugal conditions have been used in various recent studies. In order to get the optimum conditions of centrifugation for DNA-SIP, centrifugation time (36, 42, 48, 60 h), speed (45,000, 55,000 rpm) and the initial buoyant density (1.69, 1.71, 1.725 g ml-1), as were used extensively in related studies, were tested in this experiment with the Vti 65.2 rotor. DNA with either 13C-labelling or unlabelled was extracted from a paddy soil pre-incubated with either 13C-labelled or natural abundance glucose. After ultracentrifugation, the gene abundance of bacterial 16S rRNA, fungal 18S rRNA, bacterial and archaeal amoA within the fractioned DNA was detected. The results showed that centrifugation for 48 h was enough for the DNA to reach stabilization in the CsCl solution. The initial density of the mixed solution was best adjusted to 1.71 g ml-1 to ensure that most of the genes were concentrated on the middle fractions of the density gradient. Increasing the centrifugation speed would increase the density gradient of fractions; therefore, 45,000 rpm (184,000 g) was recommended so as to obtain the more widespread pattern of DNA in the centrifugal tube. We hope these findings will assist future researchers to conduct optimum ultracentrifugation for DNA-SIP.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Wang J,Zhang X,Yao Hdoi
10.1016/j.mimet.2020.105938subject
Has Abstractpub_date
2020-06-01 00:00:00pages
105938eissn
0167-7012issn
1872-8359pii
S0167-7012(19)31101-7journal_volume
173pub_type
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