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-V4 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-V4 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

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-V4 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. 70400 (24 preps)	Cat. 70410, 70420, 70430, 70440 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V3-V4 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

Propargyl-PEG4-methylamine is a PEG linker that can be react with azide compounds in copper catalyzed azide-alkyne Click Chemistry reactions. The methylamine group is reactive with carboxylic acids, activated NHS esters, carbonyls (ketone, aldehyde) etc. The PEG spacer increases hydrophilicity of the linker in aqueous environment. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.

Propargyl-PEG4-methylamine is a PEG linker that can be react with azide compounds in copper catalyzed azide-alkyne Click Chemistry reactions. The methylamine group is reactive with carboxylic acids, activated NHS esters, carbonyls (ketone, aldehyde) etc. The PEG spacer increases hydrophilicity of the linker in aqueous environment. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.

Introduction

The HiPure Plant RNA Midi Kit provides fast purification of high-quality RNA from plants, cell, tissues, and yeast using silica-membrane spin columns with a binding capacity of 1mg RNA. There is no need for phenol / chloroform extractions and time-consuming steps such as CsCl gradient ultracentrifugation or precipitation with isopropanol or LiCl. RNA purified using the RaPure Total RNA Purification System can be used for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in vitro translation.

Details

Specifications

Features	Specifications
Main Functions	Isolation total RNA from <1g simple plant sample without chloroform
Applications	RT-PCR, qPCR, Northern hybridization, second generation sequencing, nucleic acid protection, in vitro translation
Purification method	Midi spin column
Purification technology	Silica technology, DNA filtration technology
Process method	Manual (centrifugation or vacuum)
Sample type	Plant leaves, roots, stems, fruits, fungi, etc.
Sample amount	0.5-1 g
Elution volume	≥400μl
Time per run	≤40 minutes
Liquid carrying volume per column	4ml
Binding yield of column	1mg

Principle

The Kit isolates total RNA from up to 1g plant tissue. A short workflow enables RNA isolation with genomic DNA removal in less than 40 min. Samples are first lysed and homogenized. The lysate is passed through a DNA Mini column, ethanol is added to the flow-through, and the sample is applied to a RNA column. RNA binds to the membrane and contaminants are washed away. High-quality RNA is eluted in as little as 400µl water using the Kit.

Advantages

• Efficient removal of DNA – unique genomic DNA removal column without DNase treatment
• High quality – high purity total RNA can be directly used in various sensitive downstream applications
• Fast – medium isolation of several samples can be completed in 40 minutes
• Safe – no phenol chloroform extraction required
• Sensitive – RNA can be recovered at the level of PG
• Broad spectrum – various types of plant samples can be processed by diversity of operating procedures

Kit Contents

Contents	R415202	D415203
Purification Times	20 Preps	100 Preps
HiPure DNA Midi Column II	20	100
HiPure RNA Midi Columns II	20	100
15ml Collection Tubes	40	200
Buffer RLC	120 ml	550 ml
Buffer PRC1	120 ml	550 ml
Buffer RW1	70 ml	400 ml
Buffer RW2*	20 ml	100 ml
RNase Free Water	30 ml	120 ml

Storage and Stability

The Kit can be stored dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions. Make sure that all buffers are at room temperature when used. During shipment, crystals or precipitation may form in the Buffer RLC/PRC1. Dissolve by warming buffer to 37°C.

Purchase Guide

1. When dealing with woody or uncommon samples, R4150 is recommended first. R4150 contains two polysaccharide/polyphenol lysis buffer, which is the most universal product.

2. R4151 is recommended for handling common economic crop samples for the first time. Strong lysis solution can be used to process easy-extraction samples. The amount of corn or rice leaves samples can reach up to 300mg.

3. R4165 adopts CTAB/chloroform method, which can also handle a large number of difficult-to-extraction plants, but requires contact with chloroform substitutes, which is less safe than other kits. This kit uses DNase Ⅰ to remove DNA, which is also a good choice for extracting polysaccharide/polyphenol-rich plant samples.

4. R4014 is recommended for fruit/starch plant samples, which uses improved trizol pre-treatment, single column operation and is more economical.

Select the right purification kit to get impactful results：

Ⅰ. Purchase guide of Magen kits based on sample

Ⅱ. Cross purchase guide for Magen kits vs Other brands

For any customized product or OEM service, please contact us.

The HiPure Plant RNA Midi Kit provides fast purification of high-quality RNA from plants, cell, tissues, and yeast using silica-membrane spin columns with a binding capacity of 1mg RNA. There is no need for phenol / chloroform extractions and time-consuming steps such as CsCl gradient ultracentrifugation or precipitation with isopropanol or LiCl. RNA purified using the RaPure Total RNA Purification System can be used for applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA) purification, nuclease protection, and in vitro translation.