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

• Sequentially purify total RNA and total proteins from a single sample
• Kit includes a gDNA elimination column  
• No sample splitting required
• No phenol step required for efficient isolation
• Ideal for small or difficult to obtain samples
• Purify RNA and proteins from cultured animal cells, tissues, blood, bacteria, yeast, fungi or plants
• Rapid and efficient spin column procedure
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix

This kit provides a rapid, single column method for the isolation and purification of total RNA (including miRNA) and proteins sequentially from a single sample of cultured animal cells, tissues, blood, bacteria, yeast, fungi or plants. The total RNA and proteins are both column purified in under 25 minutes using a single column.

Purified RNA is of a high quality and yield, and is suitable for NGS, RT-qPCR and microarrays.  

This kit eliminates DNA efficiently using a gDNA removal column.

Proteins are eluted in buffer and are ready for downstream applications such as Western Blots, Mass Spec and ELISA.  The proteins will not require precipitation, resuspending of pellets, or any further cleaning.

This kit is ideal for researchers who are interested in studying the transcriptome and proteome of a single sample, such as for studies of microRNA profiling, gene expression including gene silencing experiments or mRNA knockdowns, studies involving biomarker discovery, and for characterization of cultured cell lines. Norgen’s RNA/Protein Purification Plus Kit is especially useful for researchers who are isolating macromolecules from precious, difficult to obtain or small samples such as biopsy materials or single foci from cell cultures, as it eliminates the need to fractionate the sample. Furthermore, analysis will be more reliable since the RNA and proteins are derived from the same sample, thereby eliminating inconsistent results. The purified macromolecules are of the highest purity and can be used in a number of different downstream applications.

Protocol

Details

Supporting Data

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Kit Specifications
Maximum Column Binding Capacity	50 μg for RNA
Maximum Column Binding Capacity	200 μg for Protein
Maximum Column Loading Volume	650 μL
Size of RNA Purified	All sizes, including small RNA (< 200 nt)
Time to Complete 10 Purifications	30 minutes
Average Yields*: HEK 293 Cells (1 x 106 cells) HEK 293 Cells (1 x 106 cells) Liver (15 mg) Liver (15 mg)	10-15 μg RNA 70-100 μg protein 30-35 μg RNA 100-150 μg protein

* Average yields will vary depending upon a number of factors including species, growth conditions used and developmental stage.

Storage Conditions and Product Stability
The Protein Loading Dye should be stored at -20°C after the addition of DL-Dithiothreitol (DTT). All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 1 year after the date of shipment.

Component	Cat. 48200 (50 preps)
Buffer SKP	40 mL
Wash Solution A	38 mL
Elution Solution A	6 mL
Wash Solution C	30 mL
Binding Buffer A	8 mL
Elution Buffer C	4 mL
Protein Neutralizer	4 mL
Protein Loading Dye	2 mL
gDNA Removal Columns	50
RNA/Protein Purification Columns	50
Collection Tubes	150
Elution Tubes (1.7 mL)	100
Product Insert	1

Introduction

HiPure Gel Pure DNA Kit uses proprietary chemistry and HiPure technology to recover DNA fragments between 80bp-15kbp with yields exceeding 80%. DNA is suitable for ligations, PCR, sequencing, restriction digestion, or various labeling reactions. In addition, this kit can be also used to recover DNA directly from enzymatic reactions such as PCR and enzyme digestion reactions.

Note: This kit can only recover 200mg of gel at most. If it is larger than 200mg, the column may be blocked. This column can only recover 5μg of DNA at most.

Details

Specifications

Features	Specifications
Main Functions	Recover DNA fragments >100bp from agarose gel(<0.2g), purification of DNA from PCR or enzymatic reaction solution solution
Applications	PCR, NGS, labeling, ligation and enzyme digestion, etc.
Purification method	Micro spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Agarose gel, PCR products, enzyme products
Sample amount	Agarose gel: ≤200mgDNA products: ≤300µl (≤5µg)
Recovery	100-5000bp：85%, 5-10kb：60-70%
Elution volume	≥10μl
Time per run	≤30 minutes（1-24 samples）
Liquid carrying volume per column	800µl
Binding yield of column	20µg

Principle

The HiPure system uses a simple bind-wash-elute procedure. Gel slices are dissolved in a buffer containing a pH indicator, allowing easy determination of the optimal pH for DNA binding, and the mixture is applied to the column. Nucleic acids adsorb to the silica-gel membrane in the high-salt conditions provided by the buffer. Impurities are washed away and pure DNA is eluted with a small volume of low-salt buffer provided or water, ready to use in subsequent applications.

Advantages

• High recovery efficiency – up to 80% DNA recovery
• Low elution volume – elution volume can be as low as 10μl
• General – recover DNA fromgel or enzyme-driven reaction solutions such as PCR
• Fast – isolation can be completed in 10-15 minutes by column gel method

Kit Contents

Contents	D211002	D211003
Purification Times	100 Preps	250 Preps
Buffer GDP	60 ml	125 ml
Buffer DW1	40 ml	90 ml
Buffer DW2	20 ml	50 ml
Elution Buffer	20 ml	30 ml
HiPure DNA Micro Columns	100	250
2 ml Collection Tubes	100	250

Storage and Stability

The Kit should be stored dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions. If any precipitates form in the buffers, warm at 37℃ to dissolve

HiPure Gel Pure DNA Kit uses proprietary chemistry and HiPure technology to recover DNA fragments between 80bp-15kbp with yields exceeding 80%. DNA is suitable for ligations, PCR, sequencing, restriction digestion, or various labeling reactions. In addition, this kit can be also used to recover DNA directly from enzymatic reactions such as PCR and enzyme digestion reactions.

Note: This kit can only recover 200mg of gel at most. If it is larger than 200mg, the column may be blocked. This column can only recover 5μg of DNA at most.