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Optimizing ultracentrifugation conditions for DNA-based stable isotope probing (DNA-SIP).
Journal of Microbiological Methods ( IF 1.7 ) Pub Date : 2020-04-29 , DOI: 10.1016/j.mimet.2020.105938
Juan Wang 1 , Xian Zhang 2 , Huaiying Yao 3
Affiliation  

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.

中文翻译:

优化基于DNA的稳定同位素探测(DNA-SIP)的超速离心条件。

DNA-SIP(基于DNA的稳定同位素探测)正越来越多地用于土壤微生物生态学,以鉴定与13C / 15N标记的底物同化的微生物。等渗梯度离心是进行DNA-SIP的主要实验过程。但是,最近的各种研究中使用了各种各样的离心条件。为了获得DNA-SIP的最佳离心条件,离心时间(36、42、48、60 h),速度(45,000、55,000 rpm)和初始浮力密度(1.69、1.71、1.725 g ml-1)在相关研究中已广泛使用,在本实验中使用Vti 65.2转子进行了测试。从已与13C标记或自然丰度葡萄糖预孵育的水稻土中提取具有13C标记或未标记的DNA。超速离心后,细菌16S rRNA的基因丰富,检测到分离的DNA中有真菌18S rRNA,细菌和古细菌amoA。结果表明,离心48小时足以使DNA在CsCl溶液中达到稳定。最好将混合溶液的初始密度调整为1.71 g ml-1,以确保大多数基因集中在密度梯度的中间部分。增加离心速度会增加馏分的密度梯度;因此,建议使用45,000 rpm(184,000 g),以便在离心管中获得更广泛的DNA模式。我们希望这些发现将有助于未来的研究人员对DNA-SIP进行最佳的超速离心。结果表明,离心48小时足以使DNA在CsCl溶液中达到稳定。最好将混合溶液的初始密度调整为1.71 g ml-1,以确保大多数基因集中在密度梯度的中间部分。增加离心速度会增加馏分的密度梯度;因此,建议使用45,000 rpm(184,000 g),以便在离心管中获得更广泛的DNA模式。我们希望这些发现将有助于未来的研究人员对DNA-SIP进行最佳的超速离心。结果表明,离心48小时足以使DNA在CsCl溶液中达到稳定。最好将混合溶液的初始密度调整为1.71 g ml-1,以确保大多数基因都集中在密度梯度的中间部分。增加离心速度会增加馏分的密度梯度;因此,建议使用45,000 rpm(184,000 g),以便在离心管中获得更广泛的DNA模式。我们希望这些发现将有助于未来的研究人员对DNA-SIP进行最佳的超速离心。增加离心速度会增加馏分的密度梯度;因此,建议使用45,000 rpm(184,000 g),以便在离心管中获得更广泛的DNA模式。我们希望这些发现将有助于未来的研究人员对DNA-SIP进行最佳的超速离心。增加离心速度会增加馏分的密度梯度;因此,建议使用45,000 rpm(184,000 g),以便在离心管中获得更广泛的DNA模式。我们希望这些发现将有助于未来的研究人员对DNA-SIP进行最佳的超速离心。
更新日期:2020-04-29
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