The 16S (V3–V4) Bacterial Amplicon Library Preparation Kit utilizes high-throughput sequencing technology to amplify the V3–V4 hypervariable region of the bacterial 16S rDNA. Combined with bioinformatics analysis, this approach enables the detection and identification of bacteria in specific environments, providing comprehensive information on species classification, relative abundance, community structure, phylogenetic relationships, and more. This kit includes all reagents required for amplification of bacterial 16S rDNA. The included 16S forward and reverse primers (F/R) are designed to amplify approximately the first 500 bp of the 16S rDNA gene. To generate Illumina-compatible 16S bacterial libraries, pair this kit with the Hieff NGS™ Tagment Index Kit for Illumina (Cat. No. 17265). All reagents provided in this kit have undergone rigorous quality control and functional validation to ensure maximum stability and reproducibility during library preparation. The 16S rRNA gene is located in the small subunit of the prokaryotic ribosome and consists of 10 conserved regions and 9 hypervariable regions (V1–V9). The conserved regions exhibit minimal sequence variation across bacterial taxa and serve as ideal binding sites for universal PCR primers, while the hypervariable regions display genus- or species-specific differences that correlate with phylogenetic distance. Therefore, the 16S rDNA gene is widely regarded as the most suitable molecular marker for bacterial phylogeny and taxonomic classification. In 16S rDNA amplicon sequencing, one or more hypervariable regions (commonly V3–V4) are selectively amplified using primers targeting the flanking conserved regions, followed by high-throughput sequencing and bioinformatic analysis of the variable regions for microbial identification. This method has become a cornerstone technique for characterizing microbial community composition in environmental samples. Amplification of the V3–V4 region of 16S rDNA from DNA extracted from diverse environmental sources allows discrimination and identification of the vast majority of bacterial species, making it the most widely adopted standard for bacterial classification in microbiome research. Features Broad Sample Compatibility: Supports metagenomic samples ranging from 100 pg to 10 ng, including feces, soil, and extracted meta-DNA. High Amplification Efficiency: Validated across multiple samples, delivering excellent library yields and high-quality sequencing data. Consistent Quality: Rigorous batch-to-batch performance and stability controls ensure reliable results. Components Components No. Name 12983ES08 (8 T) 12983ES20 (24 T) 12983ES96 (96 T) 12983-A 2×Hieff™ Multiplex PCR Master Mix 100 μL 300 μL 1200 μL 12983-B 16S Primer Mix 16 μL 48 μL 192 μL 12983-C 2×Hieff Canace™ Amplification Mix 100 μL 300 μL 1200 μL [Note]: The components in this kit are designed for the Illumina sequencing platform and must be used in conjunction with the Illumina-specific Hieff NGS™ Tagment Index Kit for Illumina (Cat# 17265) Application Microbiome Research; Microbial Community Analysis; Environmental Surveillance, Clinical Microbiology; Agriculture-related Microbial Investigations. Figures 1.Workflow Figure 1. Amplicon Library Preparation Workflow 2. High Amplification Efficiency Figure 2. Amplification performance of the 16S (V3-V4) amplicon library. Libraries were prepared using the 16S（V3-V4）Bacterial AmpSeq Prep Kit （Cat#12983） protocol for 1 ng and 10 ng of fecal, soil genomic DNA and DNA standard(the ZymoBIOMICS Microbial Community DNA Standard (Zymo Research #D6306). The results showed that Cat#12983 had a higher library yield than products from other suppliers starting input amounts (1 ng, 10 ng). 3. Excellent data performance Figure 3. Analysis of detection ratio of different microorganisms The 16S sequencing results showed that the detection of specific microbial gDNA was consistent with the expected composition. and detection performance of the kit（Cat#12983) is better than supplier T*.
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