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 V2-V3 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V2-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 V2-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 V2-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 V2-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. 70300 (24 preps)	Cat. 70310, 70320, 70330, 70340 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V2-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

Diazo Biotin-PEG3-alkyne is useful for introducing a biotin moiety to azide-containing biomolecules using Cu(I)-catalyzed Click Chemistry. The hydrophilic spacer arm provides better solubility to the labeled molecules in aqueous media. Diazo allows efficient release of captured biotinylated molecules from streptavidin using sodium dithionite (Na2S2O4). Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.

Diazo Biotin-PEG3-alkyne is useful for introducing a biotin moiety to azide-containing biomolecules using Cu(I)-catalyzed Click Chemistry. The hydrophilic spacer arm provides better solubility to the labeled molecules in aqueous media. Diazo allows efficient release of captured biotinylated molecules from streptavidin using sodium dithionite (Na2S2O4). Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.

Introduction

The Kit is specially designed for simultaneous purification of genomic DNA and total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections. Purified DNA/RNA is suitable for use in applications such as real-time PCR and pyrosequencing.

Details

Specifications

Features	Specifications
Main Functions	Co-isolation DNA and RNA from FFPE tissue
Applications	RT-PCR, cDNA synthesis, PCR and second-generation sequencing, etc.
Purification method	Polydisperse silicon based magnetic beads (DNA)Monodisperse carbonyl magnetic beads (RNA)
Purification technology	Magnetic beads technology
Process method	Manual or automatic
Adaptive instrument	Nucleic acid extractor and pipetting workstation
Sample type	FFPE slice, FFPE puncture sample, embedded tissue
Sample amount	No more than six 10 µm sections of 150 mm2 surface area or three 20µm sections of 150 mm2 surface area
Yield	DNA: 1 – 10 μg,  RNA: 1 – 25 μg

Principle

The sample is lysed and digested under the action of lysate and protease. DNA/RNA is released into the lysate. After adding magnetic particles and binding solution, DNA will be adsorbed on the surface of magnetic particles, the supernatant contain RNA was collected and bind to MagBind Particles. The adsorbed particles were washed with washing solution to remove proteins and impurities, washed with ethanol to remove salts, and finally DNA/RNA was eluted by Elution Buffer.

Advantages

• High quality – high purity total RNA / DNA can be directly used in various downstream applications
• Safe – no phenol chloroform extraction required
• Simultaneous extraction – simultaneously isolate DNA and RNA from one sample
• Post digestion sorting – higher DNA and RNA yields
• High RNA yield – monodisperse carbonyl adsorption

Kit Contents

Contents	R632701	R632702	R632703
Purification Times	48 Preps	96 Preps	5 x 96 Preps
MagBind Particles	1.1 ml	2.5 ml	11 ml
MagPure Particles N	1.1 ml	2.5 ml	11 ml
Proteinase K	24 mg	48 mg	220 mg
Protease Dissolve Buffer	3 ml	10 ml	15 ml
Buffer DPS	50 ml	100 ml	2 x 250 ml
Buffer ATL	20 ml	30 ml	120 ml
Buffer BST1	20 ml	40 ml	200 ml
Buffer BXW1*	44 ml	110 ml	3 x 110 ml
RNase Free Water	15 ml	30 ml	120 ml

Storage and Stability

Proteinase K, MagPure Particles N and MagBind Particles should be stored at 2–8°C upon arrival. However, short-term storage (up to 12 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 stablefor at least 18 months under these conditions.

Experiment Data

The Kit is specially designed for simultaneous purification of genomic DNA and total RNA from formalin-fixed, paraffin-embedded (FFPE) tissue sections. Purified DNA/RNA is suitable for use in applications such as real-time PCR and pyrosequencing.