Opentrons Flex Robot

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-V2 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V1-V2 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-V2 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-V2 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-V2 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. 70100 (24 preps)	Cat. 70110, 70120, 70130, 70140 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V1-V2 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

Overview

• Ensure optimal results for sensitive applications like NGS
• Up to a tenfold increase in microRNA mapping during sequencing runs to reduce costs
• The purified exosomal RNA is free from any circulating RNA-binding proteins.
• No phenol extractions, Proteinase K treatment, nor carrier RNA.
• No time-consuming ultracentrifugation, filtration nor special syringes are required.
• Concentrate isolated RNA into a flexible elution volume ranging from 50 μL to 100 μL.
• Purification is based on EXTRAClean spin column chromatography that uses Norgen’s proprietary separation matrix.

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.

Details

Supporting Data

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Kit Specifications (Spin Column)
Minimum Plasma Input	50 uL
Maximum Plasma Input	1 mL
Size of RNA Purified	All sizes, including miRNA and small RNA (< 200 nt)
Elution Volume	50-100 μL
Time to Complete 10 Purifications	35-40 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.