DNA extraction and 16S rRNA gene sequencing

SO Suwen Ou
HC Haipeng Chen
HW Hufei Wang
JY Jinhua Ye
HL Huidi Liu
YT Yangbao Tao
SR Songlin Ran
XM Xiaoqin Mu
FL Fangzhou Liu
SZ Shuang Zhu
KL Kangjia Luo
ZG Zilong Guan
YJ Yinghu Jin
RH Rui Huang
YS Yanni Song
SL Shu-lin Liu
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Genomic DNA (gDNA) was extracted from fresh frozen colorectal cancer tissue with the E.Z.N.A.® tissue DNA Kit (Omega Bio-tek, U.S.) and from fecal samples with the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, U.S.) according to manufacturer’s instructions. The V3-V4 regions of the 16S rRNA gene were PCR amplified using primer pairs 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′ GGACTACHVGGGTWTCTAAT-3′). The PCR reaction was set up in triplicate, and the PCR product was purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA). The high-throughput sequencing was performed in Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). Analysis of the 16S rRNA microbiome sequencing data was performed using the free online platform of Majorbio Cloud Platform (cloud.majorbio.com).

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