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 FunctionsC	Co-isolation total RNA and DNA from FFPE tissue
Applications	RT-PCR, cDNA synthesis, PCR and second-generation sequencing, etc.
Purification method	Polydisperse magnetic beads
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 10µm sections of 150 mm2 surface area or three 20µm sections of 150 mm2 surface area.

Principle

FFPE samples are incubated in an optimized lysis buffer, which results in the release of RNA and precipitation of DNA. After centrifugation, the RNA-containing supernatant and DNA-containing pellet are then processed separately to purify RNA and DNA. After adding magnetic particles and binding solution, DNA/RNA 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/RNA was eluted by RNase Free Water.

Advantages

• Post digestion sorting – higher DNA and RNA yields
• Economy – the price is much lower than imported reagents

Kit Contents

Contents	IVD3026
Purification Times	200 Preps
MagBind Particles	9.0 ml
Proteinase K	180 mg
Protease Dissolve Buffer	10 ml
Buffer DPS	150 ml
Buffer FRL	40 ml
Buffer ATL	40 ml
Buffer AL	80 ml
Buffer BXW1*	110 ml
RNase Free Water	30 ml

Storage and Stability

Proteinase K and MagBind Particles should be stored at 2-8°C upon arrival. However, short-term storage (up to 24 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 stable for at least 18 months under these conditions.

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.

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

2-(Propargyl-PEG4-amido)-1,3bis(PEG1-methyl ester) is a crosslinker that can react with azide compounds or biomolecules via copper catalyzed Click Chemistry to form a stable triazole linkage. The methyl ester groups can be hydrolyzed, reduced, or substituted under different conditions.

2-(Propargyl-PEG4-amido)-1,3bis(PEG1-methyl ester) is a crosslinker that can react with azide compounds or biomolecules via copper catalyzed Click Chemistry to form a stable triazole linkage. The methyl ester groups can be hydrolyzed, reduced, or substituted under different conditions.