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

Overview

• Isolate DNA from a wide range of food materials. (e.g boiled, fluid, processed or raw food products)
• No hazardous chemicals required (e.g. phenol or chloroform)
• Effective lysis with Proteinase K and optional lysozyme treatment
• Fast (less than 15 minutes hands-on time) and convenient processing using a rapid spin-column format
• Wide compatibility with a variety of food products for GMO-DNA isolation
• Universal protocol for food related pathogen DNA isolation (Gram positive and Gram negative)

This kit provides a rapid spin column method for the isolation and purification of total DNA from a wide range of food samples originating from animals or plants. The kit is designed for identification of GMO-DNA or animal components in food and feed and can be used for a wide range of starting materials including raw or processed food, meat, liquids, sauces and dairy products including milk, cheese and yogurt.

This kit also provides a convenient method for the detection of food-related pathogens and will isolate such DNA (enriched or as is) including Gram-positive bacteria, Gram-negative bacteria, yeast and fungi which may contaminate food sources.  A number of pathogens have been tested including E. coli O157:H7, Staphylococcus, Listeria monocytogenes, Salmonella enterica & Campylobacter jejuni.  The purified DNA is of the highest integrity, and can be used in a number of downstream applications including PCR based detection, sequencing and genotyping.  

Details

Supporting Data

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Kit Specifications
Maximum Column Binding Capacity	50 μg
Maximum Column Loading Volume	650 μL
Maximum Amount of Starting Material: Solid food material Liquid sample (e.g. milk or concentrated juice)	200 mg 1 mL to 1.5 mL
Time to Complete 10 Purifications	45 minutes

Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 1 year after the date of shipment. The kit contains a ready-to-use Proteinase K, which is dissolved in a specially prepared storage buffer. The buffered Proteinase K is stable for up to 1 year after the date of shipment when stored at room temperature.

Samples Tested by PCR

Food Materials	Samples that Have Been Tested by PCR
Plant related	Cereal, Jam, Chocolate, Spices, Sauce
Animal related	Raw and processed meat products (e.g. ham, beef jerky, taco seasoned ground beef, pork and sausage)
Dairy product	Milk, Yogurt, Cheese
Pathogens (enriched from food samples)	E. coli O157:H7 from food sample Staphylococcus from milk Listeria monocytogenes from milk Salmonella enterica from raw meat Campylobacter jejuni from milk

Component	Cat. 54500 (50 preps)
Lysis Buffer L	60 mL
Binding Buffer I	7 mL
Buffer SK	30 mL
Wash Solution A	18 mL
Elution Buffer B	8 mL
Proteinase K	1 vial
Spin Columns	50
Collection Tubes	50
Elution Tubes (1.7 mL)	50
Product Insert	1

Introduction

This product is suitable for rapid extraction of DNA from tissue, cells, blood, saliva, swabs, blood spots, semen and other clinical samples. DNA can be used directly for PCR, quantitative PCR, Southern Blot, test of virus DNA and so on.

Details

Specifications

Features	Specifications
Main Functions	Isolation total DNA from 1-100ul blood, FFPE, tissue and other samples
Applications	Second generation sequencing, PCR, real timePCR, etc.
Purification method	Monodisperse magnetic beads
Purification technology	Magnetic beads technology
Process method	Manual or automatic
Sample type	Anticoagulant blood, concentrated blood, buffy coat, lymphocytes and cultured cells
Sample amount	Body fluid : 10 – 200μl, Tissue : ≤10mg
Yield	10ng – 15μg
Elution volume	80 -100μl
Time per run

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

• High binding force – suitable for handling DNA rich samples, such as whole blood, buffy coat, concentrated blood, etc.
• High purity – carboxyl magnetic beads with weak surface adsorption, getting higher purity
• General – can be used for various clinical samples

Kit Contents

Contents	IVD3101
Purification Times	200
MagBind Particles	4.5 ml
Proteinase K	90 mg
Protease Dissolve Buffer	10 ml
Buffer ATL	60 ml
Buffer AL	60 ml
Buffer BD*	20 ml
Buffer BXW1*	110 ml
Elution Buffer	30 ml

Storage and Stability

Proteinase K, MagBind Particles should be stored at 2–8°C upon arrival. However, short-termstorage (up to 18 weeks) at room temperature (15–25°C) does not affect their performance. The remainingkit components can be stored dry at room temperature (15–25°C) and are stable for at least 18 months under these conditions.

This product is suitable for rapid extraction of DNA from tissue, cells, blood, saliva, swabs, blood spots, semen and other clinical samples. DNA can be used directly for PCR, quantitative PCR, Southern Blot, test of virus DNA and so on.