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

Introduction

HiPure RNA Clean Up Kit provides a rapid and easy method for the purification and concentrate RNA from enzymatic reactions or for desalting the RNA samples. RNA purified using HiPure RNA Clean Up Kit is ready for all downstream applications such as RT-PCR, Northern blotting, mRNA purification, nuclease protection, and in vitro translation.

Details

Specifications

Features	Specifications
Main Functions	Purification and concentration of RNA（mRNA>200nt or miRNA）from transcription products, DNase cleavage products, labeled products, and crude RNA products
Applications	Sequencing, ligation, enzyme digestion, RT-PCR, labeling, etc.
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Crude RNA, enzymatic reaction solution
Sample amount	≤100μl
Recovery	90%
Elution volume	≥15μl
Time per run	≤15 minutes
Liquid carrying volume per column	800μl
Binding yield of column	100μg

Principle

The HiPure system uses a simple bind-wash-elute procedure. Binding buffer is added directly to the sample or other enzymatic reaction, and the mixture is applied to the column. Nucleic acids adsorb to the silica membrane in the high-salt conditions provided by the buffer. Impurities are washed away and pure RNA is eluted with a small volume of low-salt buffer provided or water, ready to use in all subsequent applications.

Advantages

• High recovery – recovery of RNA up to 90%
• Low elution volume – the elution volume can be as low as 15µl
• Fast – only 10 minutes for recovery by using column method
• General – suitable for various crude RNA products
• Wide range of fragment recovery:(>25nt RNA)

Kit Contents

Contents	R214402	R214403
Purification Times	50 Preps	250 Preps
Buffer GXP	30 ml	120 ml
Buffer RW2	20 ml	2 x 50 ml
RNase-Free Water	20 ml	60 ml
HiPure RNA Mini Columns I	50	250
2 ml Collection Tubes	50	250

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. The entire kit can be stored 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.

HiPure RNA Clean Up Kit provides a rapid and easy method for the purification and concentrate RNA from enzymatic reactions or for desalting the RNA samples. RNA purified using HiPure RNA Clean Up Kit is ready for all downstream applications such as RT-PCR, Northern blotting, mRNA purification, nuclease protection, and in vitro translation.

About

PACE Multiplex Master Mix for the simultaneous detection of up to four targets in one reaction. Save time, cost, and consumables while maximising data generation.

PACE Multiplex Master Mix is an advanced and versatile extension of our PACE 2.0 Genotyping Master Mix, formulated for the simultaneous detection of up to four targets in one reaction well. For example, two bi-allelic SNPs, or one reference gene and a further three genes of interest.

PACE Multiplex genotyping assay designs are available from 3CR Bioscience through our free PACE assay design service; once designed, users can purchase assay primers independently or through 3CR Bioscience using our partial or full-assay validation service.

Users will require a plate reader capable of reading FAM, HEX, ATTO 590, ATTO 647N and reference dye ATTO 680 (wavelengths in the PACE Multiplex Master Mix User Guide). PACE Multiplex Master Mix is supplied at 2x concentration for convenience and with or without ATTO 680 reference dye at a range of levels to ensure compatibility with your qPCR machine or reader.

PACE Multiplex Master Mix for the simultaneous detection of up to four targets in one reaction. Save time, cost, and consumables while maximising data generation.