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

Figure 1 / 3

Previous

Next

Click for expanded view

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

Introduction

Free-circulating nucleic acids, such as tumor-specific extracellular DNA fragments and mRNAs in the blood or fetal nucleic acids in maternal blood, are present in serum or plasma usually as short fragments, <1000bp (DNA). HiPure Circulating DNA Midi Kit enables efficient purification of these circulating nucleic acids from human plasma, serum, or urine.

This product is for research use only.

Video

Details

Specifications

Features	Specifications
Main Functions	Isolation circulating DNA from 1-5ml plasma, serum, body fluids using vacuum protocol
Applications	qPCR, liquid or solid chip analysis, hybridization and SNP detection, etc.
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (vacuum)
Sample type	Serum, plasma and other cell-free fluid samples
Sample amount	1-5ml
Elution volume	≥50μl
Time per run	≤60 minutes
Liquid carrying volume per column	4ml
Binding yield of column	1mg

Principle

This product is based on silica Column purification. The sample is lysed and digested with lysate and protease, DNA is released into the lysate. Transfer to an adsorption column. Nucleic acid is adsorbed on the membrane, while protein is not adsorbed and is removed with filtration. After washing proteins and other impurities, Nucleic acid was finally eluted with low-salt buffer.

Advantages

• High yield – most optimal process, free DNA (>50bp) can be obtained to the maximum extent
• High concentration – low elution volume, ensuring high nucleic acid concentration
• High purity – low alcohol binding method, completely removing inhibitor and protein pollution
• High recovery – DNA can be recovered at thelevel of PG by silica gel column purification

Kit Contents

Contents	D318201C	D318202C
Purification Times	10	50
Buffer ACL	50 ml	250 ml
Buffer ACB*	60 ml	300 ml
Buffer DCW1*	4.4 ml	22 ml
Buffer DCW2*	5 ml	10 ml
Proteinase K	110 ml	540 mg
Protease Dissolve Buffer	10 ml	30 ml
Carrier RNA	110 μg	110 μg
Nuclease Free Water	10 ml	20 ml
HiPure CFDNA Mini Columns	10	50
2 ml Collection Tubes	20	100
Extender Tube	10	50
Vac-Connector	10	50

Storage and Stability

Proteinase K, Carrier RNA 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 dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions. The entire kit can be stored at 2-8°C, but in this case buffers should be redissolved before use. Make sure that all buffers are at room temperature when used.

Experiment Data

Free-circulating nucleic acids, such as tumor-specific extracellular DNA fragments and mRNAs in the blood or fetal nucleic acids in maternal blood, are present in serum or plasma usually as short fragments,

Content:	100 assays per kit
Shipping Temperature:	Ambient
Storage Temperature:	Short term stability: 2-8oC, Long term stability: See individual component labels
Stability:	> 2 years under recommended storage conditions

Analyte:	Histamine
Assay Format:	Spectrophotometer
Detection Method:	Absorbance
Wavelength (nm):	492
Signal Response:	Increase
Linear Range:	0.5 to 12 μg of histamine per assay
Limit of Detection:	~ 0.09 μg of histamine per test or ~ 0.75 mg/Kg (ppm) of histamine in a fish sample
Limit of Quantification:	~ 0.5 mg of histamine per test or  ~ 4.2 mg/Kg (ppm) of histamine in a fish sample
Reproducibility (%):	~ 3%
Reaction Time (min):	~ 20 min
Application examples:	Fish samples in various forms (e.g. fresh, canned, frozen, etc).

This product has been discontinued.

The Histamine Assay Kit for the detection of histamine in food including fish samples in various forms.

High levels of histamine may develop in a variety of foods as they decompose, this is an issue that particulary affects fish.

Megazyme’s Histamine Assay Kit provides a simple robust method with a fast reaction time of ~ 20 mins.

View all of our assay kits.

Advantages

• Very competitive price (cost per test)
• All reagents stable for > 2 years
• Specific for histamine
• Suitable for use with raw fish samples
• Simple format. Well suited to automation
• Standard included
• Mega-Calc™ software tool is available from our website for hassle-free raw data processing

The Histamine Assay Kit for the detection of histamine in food including fish samples in various forms.