4x500ml Angle Rotor

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

Sample type purification kit guide 

The 16S V3-V5 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V3-V4 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 V3-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 V3-V4 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

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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 V3-V5Library 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. 70500 (24 preps)	Cat. 70510, 70520, 70530, 70540 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V3-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

DBCO-C5-PEG12-acid is an analog of DBCO-Acid with PEG linker and a DBCO group. DBCO group is a highly reactive cycloalkyne which is commonly used for copper-free Click Chemistry reactions. The hydrophilic PEG chain allows for increased water solubility of compounds in aqueous media. The terminal carboxylic acid can react with primary amine groups in the presence of activators (e.g. HATU) to form a stable amide bond. Reagent grade, for research use only.

DBCO-C5-PEG12-acid is an analog of DBCO-Acid with PEG linker and a DBCO group. DBCO group is a highly reactive cycloalkyne which is commonly used for copper-free Click Chemistry reactions. The hydrophilic PEG chain allows for increased water solubility of compounds in aqueous media. The terminal carboxylic acid can react with primary amine groups in the presence of activators (e.g. HATU) to form a stable amide bond. Reagent grade, for research use only.