Gel images of different ranges of library size selection. Sheared human genomic DNA was used as input.

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Library size selection is an enrichment of a specific range of library sizes for NGS library preparations. The NGS library preparation is related to the quality of the sequencing data. Precise NGS library size selection can increase sequencing efficiency, improve data quality, and reduce costs.

There are two types of sequencing technologies: short-read sequencing and long-read sequencing. Short-read sequencing uses DNA libraries that contain small insert DNA fragments of similar sizes, usually several hundred base pairs. The sequencing efficiency can be improved if the DNA size selection is in the right range. Cat.# 20104S and 20104L are the best kits for NGS library size selection of illumina paired-end 100 (PE100) sequencing with 100-200 bp library inserts; Cat.# 20105S and 20105L are the best kits for NGS library size selection of illumina paired-end 150 (PE150) sequencing with 150-300 bp library inserts; and Cat.# 20106S and 20106L are the best kits for NGS library size selection of illumina paired-end 300 (PE300) sequencing with 300-600 bp library inserts.

Long-read sequencing uses a large DNA fragment as input and makes very long reads. Usually, library size selection is preferred to remove smaller fragments. Cat.# 20110S and 20110L are the best kits for long-read sequencing size selection with DNA sizes >5 kb, and Cat.# 20111S and 20111L are the best kits for long-read sequencing size selection with DNA sizes >10 kb.

The magnetic beads, or SPRI (Solid Phase Reversible Immobilization) beads, is well used for the purification of DNA due to their reversible DNA binding. The NGS library can be size-selected by the magnetic beads or SPRI beads. The properties of the magnetic beads can be changed for a specific range of DNA binding. The contaminants and other unwanted components in the libraries can also be removed during size selection.

Specific ranges of NGS libraries can be selected using magnetic beads with different buffer compositions. The first DNA-beads binding step, also called the right-side clean-up, removes large NGS library fragments. The large NGS library fragments that bind to the beads are discarded with the beads pellet. The desired NGS library fragments in the supernatant are transferred to a new well, and new beads are added to the supernatant for the second beads-DNA binding, also called the left-side clean-up. After the rinsing step, the NGS library fragments with the dual selection are eluted in water or an appropriate buffer. The magnetic beads method has great advantages over time-consuming column purification and tedious gel-based purification.

NGS library size selection with dual clean-ups.

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Library size selection for long-read sequencing only requires a single clean-up. In this case, only the large library fragments are bound to the beads, while other small library fragments are discarded with the supernatant. The selected larger library fragments are eluted in water or an appropriate buffer after the rinsing step.

NGS library size selection with single clean-up for >5 kb and >10 kb libraries.

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Features of NGS library size selection

• • • 5 ranges for NGS library size selection
      • • illumina PE100 sequencing: 250-350 bp
        • illumina PE150 sequencing: 300-450 bp
        • illumina PE100 sequencing: 450-750 bp
        • Long-read sequencing: >5 kb
        • Long-read sequencing: >10 kb
    • Rapid protocol: only 20 minutes
    • Simple workflow
      • • No columns required
        • No gel purification required
        • No centrifugation required

Overview

• Detection kits for the HBV
• Available in TaqMan format for analysis

The Hepatitis B virus (HBV) is mainly transmitted via blood or blood products. In addition, sexual, oral and perinatal infections are also possible. Early symptoms of the infection include appetite loss, vomiting and abdominal symptoms, with approximately 10-20% of those patients developing fever as well as rheumatoid joint and muscle pain. Jaundice, which may be accompanied by itching, will then develop within 2-14 days. Fulminant hepatitis then occurs in about 1% of all infected patients, which in severe cases may be fatal. Of those individuals infected by HBV, 5-10% will develop chronic liver inflammation which may progress to cirrhosis of the liver or, in the worst case, primary liver cell carcinoma. The HBV virus is a hepadnavirus that has a circular genome composed of partially double-stranded DNA.

HBV TaqMan PCR Kit, 100 reactions

• Ready to use format, including Master Mix for the target and PCR control to monitor for PCR inhibition and validate the quality
• Specific Primer and Probe mix for the pathogen/virus/viroid of interest
• Primer and Probe mix
• Positive and negative control to confirm the integrity of the kit reagents

HBV TaqMan PCR Probe/Primer Set and Controls, 100 reactions

• Specific Primer/Probe mix and Positive Control for the pathogen/virus/viroid of interest
• Nuclease-free water
• Can be used together with Norgen’s PCR Master Mix (#28007) or customer supplied master mix

For research use only and NOT intended for in vitro diagnostics.

Details

Supporting Data

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Storage Conditions and Product Stability
All kit components can be stored for 2 years after the date of production without showing any reduction in performance.

All kit components should be stored at -20°C upon arrival. Repeated thawing and freezing (> 2 x) of the Master Mix and Positive Control should be avoided, as this may affect the performance of the assay. If the reagents are to be used only intermittently, they should be frozen in aliquots.

Component	Cat. TM29250 (100 rxns)	Cat. TM29210 (100 rxns)
MDx TaqMan 2X PCR Master Mix	2 x 700 μL	–
HBV Primer & Probe Mix	280 μL	280 μL
HBV Positive Control	150 μL	150 μL
Nuclease-Free Water (Negative Control)	1.25 mL	1.25 mL
Product Insert	1	1

Introduction

The HiPure Liquid RNA & miRNA Kit integrates phenol/guanidine-based sample lysis and silica membrane purification of total RNA. MagZol LS Reagent, included in the kits, is a monophasic solution of phenol and guanidine thiocyanate designed to facilitate lysis of liquid sample and inhibit RNase. The high lysis efficiency of the reagent and the subsequent removal of contaminants by organic phase extraction enable the use of up to 0.25ml liquid sample per mini spin column.

Details

Specifications

Features	Specifications
Main Functions	Isolation total RNA from 0.25ml body fluids using column and MagZol LS reagent
Applications	qPCR / RT-PCR, liquid or solid-phasechip analysis, hybridization and SNP detection
Purification method	Mini spin column
Purification technology	Silica technology, acidphenol / guanidine extraction technology (MagZol pretreatment technology)
Process method	Manual (centrifugation or vacuum)
Sample type	Blood, serum, plasma
Sample amount	250μl
Elution volume	≥20μl
Liquid carrying volume per column	800µl
Binding yield of column	100μg

Principle

0.25ml liquid samples are homogenized in 0.75ml MagZol LS Reagent. After addition of chloroform, the homogenate is separated into aqueous and organic phases by centrifugation. The upper, aqueous phase is extracted, and ethanol is added to provide appropriate binding conditions. The sample is then applied to the spin column, where the total RNA (up to 100 µg) binds to the membrane and phenol and other contaminants are efficiently washed away. High-quality RNA is then eluted in 30–100µl of RNase-free water.

Advantages

• High quality – high purity total RNA can be directly used in various sensitive downstream applications
• Fast – isolation of several samples can be completed in 40 minutes by using column purification method
• Safety – no phenol chloroform extraction required
• Sensitive – direct lysis of blood, plasma and other samples without separation of leukocytes

Kit Contents

Contents	R416302	R416303
Purification Times	50 Preps	250 Preps
HiPure RNA Mini Columns	50	250
2ml Collection Tubes	50	2 x 125
MagZol LS Reagent	60 ml	270 ml
Buffer RWC	20 ml	60 ml
Buffer RW2*	20 ml	2 x 50 ml
RNase Free Water	10 ml	30 ml

Storage and Stability

MagZol LS Reagent 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 dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions.

The HiPure Liquid RNA & miRNA Kit integrates phenol/guanidine-based sample lysis and silica membrane purification of total RNA. MagZol LS Reagent, included in the kits, is a monophasic solution of phenol and guanidine thiocyanate designed to facilitate lysis of liquid sample and inhibit RNase. The high lysis efficiency of the reagent and the subsequent removal of contaminants by organic phase extraction enable the use of up to 0.25ml liquid sample per mini spin column.