N-(Boc-PEG3)-N-bis-(PEG3-Amino-Tri-(Propargyl-PEG2-ethoxymethyl)-methane) is a crosslinker consisting of six propargyl groups and a t-Boc protected aminooxy group. The propargyl groups can form triazole linkage with azide-bearing compounds or biomolecules via copper catalyzed Click Chemistry. The protected amine can be deprotected under acidic conditions. The hydrophilic PEG spacer increases solubility in aqueous media. Reagent grade, for research use only.
N-(Boc-PEG3)-N-bis-(PEG3-Amino-Tri-(Propargyl-PEG2-ethoxymethyl)-methane) is a crosslinker consisting of six propargyl groups and a t-Boc protected aminooxy group. The propargyl groups can form triazole linkage with azide-bearing compounds or biomolecules via copper catalyzed Click Chemistry. The protected amine can be deprotected under acidic conditions. The hydrophilic PEG spacer increases solubility in aqueous media. Reagent grade, for research use only.
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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.
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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.
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Norgen’s EXTRAClean Plasma/Serum Exosome and Free-Circulating RNA Isolation Kit constitutes an all-in-one system for the purification of exosomes and the sequential isolation of RNA and free-circulating RNA from different plasma/serum sample volumes ranging from 50 μL and up to 10 mL. The purification is based on spin column chromatography that employs Norgen’s proprietary resin. The EXTRAClean columns undergo stringent processing and rigorous quality control measures to minimize contamination traces, ensuring optimal results for sensitive applications such as NGS. The kit is designed to isolate all sizes of RNA, including microRNA. The kit provides a clear advantage over other available kits in that they do not require any special instrumentation, protein precipitation reagents, extension tubes, phenol/chloroform or any protease treatments. Moreover, the kit allows the user to elute into a flexible elution volume ranging from 50 μL to 100 μL. The RNA isolated from the purified exosomes is free from any protein-bound circulating RNA and is of the highest integrity. Moreover, the free-circulating, protein-bound, RNA is free from any exosomal RNA. The purified RNA can be used in a number of downstream applications including real time PCR, NGS application, reverse transcription PCR, Northern blotting, RNase protection and primer extension, and expression array assays.
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| Kit Specifications (Spin Column) | |
| Minimum Plasma Input | 4 mL |
| Maximum Plasma Input | 10 mL |
| Size of RNA Purified | All sizes, including miRNA and small RNA (< 200 nt) |
| Elution Volume | 50-100 μL |
| Time to Complete 10 Purifications | 40-45 minutes |
| Average Yields | Variable depending on specimen |
Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.
