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 V1-V3 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V1-V3 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 V1-V3 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 V1-V3 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 V1-V3 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. 70200 (24 preps)	Cat. 70210, 70220, 70230, 70240 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V1-V3 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

Introduction

This kit is suitable for high-throughput extraction of total RNA from paraffin embedded tissue samples, including miRNA. The kit is based on superparamagnetic magnetic particle purification technology. The extraction process does not require the use of toxic phenolic chloroform extraction or time-consuming alcohol precipitation.The whole extraction process only takes 120 minutes. The obtained RNA can be directly used in RT-PCR. The high-throughput extraction can be realized by 96 hole magnetic frame and 96 holeo scillator, various nucleic acid extractors (such as KingFisherFle, MagMix32, MagMix 96) or cooperating with the pipette workstation (Hamilton, Tican) for automatic extraction.

Details

Specifications

Features	Specifications
Main Functions	Isolation total RNA from FFPE tissue
Applications	RT-PCR, cDNA synthesis, second generation sequencing
Products	RNA, miRNA
Purification method	Polydisperse magnetic beads
Purification technology	Magnetic beads technology
Process method	Manual or automatic
Adaptive instrument	Nucleic acid extractor, pipetting workstation
Sample type	FFPE slice, FFPE embedded tissue
Sample amount	≤6 slices

Principle

This product is based on the purification method of high binding magnetic particles. The sample is lysed and digested under the action of lysate and Protease. DNA is released into the lysate. After adding magnetic particles and binding solution, DNA will be adsorbed on the surface of magnetic particles, and impurities such as proteins will be removed without adsorption. The adsorbed particles were washed with washing solution to remove proteins and impurities, washed with ethanol to remove salts, and finally DNA was eluted by Elution Buffer.

Advantages

• Fast – several samples can be extracted in 120 minutes by column method
• Safe – no phenol chloroform extraction required

Kit Contents

Contents	IVD3022
Purification Times	200 Preps
MagBind Particles	4.5 ml
Proteinase K	100 mg
Protease Dissolve Buffer	6 ml
DNase I	4 x 600 µl
DNase Buffer	30 ml
Buffer DPS	200 ml
Buffer FRL	40 ml
Buffer AL	40 ml
Buffer MW1*	110 ml
Buffer MW2*	2 x 50 ml
Nuclease Free Water	30 ml

Storage and Stability

Proteinase K and MagBind Particles should be stored at 2–8°C upon arrival. DNase I should bestored at -20°C. However, short-term storage (DNase I up to 1 weeks, Proteinase K and MagBind Particles up to 8 weeks) at room temperature (15–25°C) does not affect their performance. The remaining kit components can be stored at room temperature (15–25°C) and are stable for at least 18 months under theseconditions.

This kit is suitable for high-throughput extraction of total RNA from paraffin embedded tissue samples, including miRNA. The kit is based on superparamagnetic magnetic particle purification technology. The extraction process does not require the use of toxic phenolic chloroform extraction or time-consuming alcohol precipitation.The whole extraction process only takes 120 minutes. The obtained RNA can be directly used in RT-PCR. The high-throughput extraction can be realized by 96 hole magnetic frame and 96 holeo scillator, various nucleic acid extractors (such as KingFisherFle, MagMix32, MagMix 96) or cooperating with the pipette workstation (Hamilton, Tican) for automatic extraction.