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 plasmausually as short fragments, <1000bp(DNA). HiPure Circulating DNA Midi Kit enables efficient purification of these circulating nucleic acids from human plasma, serum, or urine. The extracted products can be used for clinical in vitro detection.

Details

Specifications

Features	Specifications
Main Functions	Isolation circulating DNA from 1-5ml plasma, serum, bodyfluids by spin
Applications	qPCR, liquid or solid chip analysis, hybridization and SNP detection, etc.
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation)
Sample type	Serum, plasma and other cell-free fluid samples
Sample amount	1-5ml
Elution volume	≥50μl
Time per run	≤70 minutes
Liquid carrying volume per column	4ml
Binding yield of column	1mg

Principle

This product is based on silica gel purification. The sample is lysed and digested with lysate and protease, DNA is released into the lysate. Transfer to an adsorption plate and filter 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 (10 Mm Tris,pH 8.0).

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	D318203D
Purification Times	50 Preps
Buffer ACL	250 ml
Buffer ACB*	300 ml
Buffer DCW1*	22 ml
Buffer DCW2*	10 ml
Proteinase K	540 mg
Protease Dissolve Buffer	30 ml
Carrier RNA	110 μg
Nuclease Free Water	20 ml
HiPure CFDNA Mini Columns	50
2 ml Collection Tubes	100
Extender Tubes	50
Sealing O-Ringes	50
50ml Collection Tube	50
Support Tube	50

Storage and Stability

Proteinase K, Carrier RNA should be stored at 2-8°C upon arrival. However, short-term storage (up to 12weeks) 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 bestored 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.

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 plasmausually as short fragments,

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 V1-V3 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V1-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 V1-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 V1-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 V1-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. 70200 (24 preps)	Cat. 70210, 70220, 70230, 70240 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V1-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

Mal-amido-PEG4-alkyne is a short PEG linker featuring a maleimide and a terminal alkyne. Maleimide is a covalent ligand used for linking thiols such as those in cysteines residues in proteins, while terminal alkynes can be used in copper (I) click chemistry with azides on target molecules.

Mal-amido-PEG4-alkyne is a short PEG linker featuring a maleimide and a terminal alkyne. Maleimide is a covalent ligand used for linking thiols such as those in cysteines residues in proteins, while terminal alkynes can be used in copper (I) click chemistry with azides on target molecules.