16S V4-V5 Library Preparation Kit for Illumina
The reagents and components required for library preparation of the 16S V4-V5 amplicon libraries to be used for next-generation sequencing on Illumina platforms
For research use only and NOT intended for in vitro diagnostics.
16S V4-V5 Library Preparation Kit for Illumina
The reagents and components required for library preparation of the 16S V4-V5 amplicon libraries to be used for next-generation sequencing on Illumina platforms
Overview
- Protocol optimized for DNA isolated from a diversity of samples including stool, soil, water, saliva, plant, urine, skin, and more
- Simple and quick workflow: library could be prepared in less than 5 hours
- Component of Norgen's metagenomics workflow
- A single NGS run can be prepared with up to 384 unique dual-index libraries
The 16S V4-V5 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V4-V5 amplicon libraries to be used for next-generation sequencing on Illumina platforms. All molecular reagents including primers, enzyme mixes, indexes, and buffers are provided. Instructions for PCR clean up with the AMPure XP Magnetic Beads (supplied by customer) are also included for rapid purification of nucleic acid products generated at two steps of the workflow. The library prep workflow could be used for purified DNA inputs from different sources including stool, soil, water, saliva, plant, urine, skin swab, vaginal swab, cheek swab, nasal swab, plasma/serum, tongue swab, gum swab, and others.
The 16S V4-V5 Library Preparation Kit for Illumina has a streamlined procedure that reduces the handling time such that the library prep procedure can be completed in approximately 4 hours (see diagram below). Input DNA is first subjected to targeted PCR to amplify the V4-V5 region of the DNA encoding 16S rRNA. The post-PCR reaction is then cleaned up using AMPure XP beads. Dual index primers are then added using a limited-cycle PCR. The indexed amplicons flanked by 5' and 3' barcoded adaptors are then cleaned using AMPure XP beads. The libraries are then ready for quantification, pooling and sequencing.
Details
Supporting Data
Minimum amount of starting material: | 2.5 µL of DNA (5 ng/µL) |
Time to complete library preparation: | 4 hours |
Storage Conditions and Product Stability
Norgen’s 16S V4-V5 Library Prep Kit for Illumina is shipped as one kit box (for
the 24 prep kit) or two sub-component kits (for the 96 prep kit). All kits should be
stored at -20°C upon arrival.
All kit components should remain stable for at least 1 year when stored at the specified storage conditions.
Step | Component | Cat. 70700 (24 preps) | Cat. 70710, 70720, 70730, 70740 (96 preps) |
---|---|---|---|
Amplicon PCR (PCR 1) | MGX Master Mix | 330 µL | 1,320 µL |
16S V4-V5 Primer Mix | 70 µL | 280 µL | |
Index PCR (PCR 2) | Indexing Master Mix | 660 µL | 2 x 1,320 µL |
N7xx Index Primer | 50 µL | 50 µL | |
S5xx Index Primer | 70 µL | 70 µL | |
PCR Clean-Up | Resuspension Buffer | 2 x 1,250 µL | 2 x 5,000 µL |
Nuclease-free water | 1,250 µL | 1 x 6,000 µL |
Documentation
Citations
Title | Extension of bacterial rDNA sequencing for simultaneous methylation detection and its application in microflora analysis |
Citation | scientific reports 2023. |
Authors | Motoi Nishimura, Tomoaki Tanaka, Syota Murata, Akiko Miyabe, Takayuki Ishige, Kenji Kawasaki, Masataka Yokoyama, Naoko Hashimoto, Kazuyuki Yamagata, Hidekazu Nagano, Satomi Tojo-Nishimura & Kazuyuki Matsushita |