RNA Basic Isothermal PCR Kit With High Accuracy And Efficiency

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 V2-V3 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V2-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 V2-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 V2-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 V2-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. 70300 (24 preps)	Cat. 70310, 70320, 70330, 70340 (96 preps)
Amplicon PCR (PCR 1)	MGX Master Mix	330 µL	1,320 µL
	16S V2-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

Opentrons OT-2 Single-Channel Electronic Pipettes automate liquid handling and pipetting for most daily wetlab applications. Best for low to medium throughput workflows with high precision.

Opentrons OT-2 Single-Channel Electronic Pipettes automate liquid handling and pipetting for most daily wetlab applications. Best for low to medium throughput workflows with high precision.

Overview

• Concentrate circulating DNA into a flexible elution volume ranging from (25 µL – 50 µL)
• Isolate inhibitor-free cell-free circulating DNA
• Compatible with Streck Cell-Free DNA BCT® Tubes
• Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix
• Research use format is also available

These kits provide a fast, reliable and convenient spin column method for the isolation of high quality, high purity and inhibitor-free cell-free circulating DNA (cfc-DNA) from plasma/serum sample. These kits are designed to isolate all sizes of cfc-DNA from either fresh or frozen plasma/serum samples and the purified DNA is eluted into a flexible elution volume ranging from 25 µL to 50 µL. The purified plasma/serum cfc-DNA is fully compatible with all downstream applications including PCR, qPCR, methylation-sensitive PCR and Southern Blot analysis, microarrays and NGS.

Background

Plasma/Serum cell-free circulating DNA (cfc-DNA) has the potential to provide biomarkers for certain cancers and disease states as well as fetal DNA in maternal blood. Currently, significant advancements are being made in utilizing cfc-DNA as biomarkers for the early diagnosis, prognosis and monitoring of therapy for several cancer types and autoimmune diseases. Cell-free mitocondrial DNA (cfmtDNA) is also under investigation for its clinical significance. This cfc-DNA is usually present as short fragments of less than 1000 bp. In addition, cell-free fetal DNA has been widely used as a non-invasive method for prenatal diagnosis including early identification of fetal sex, genetic studies for families at high risk for inherited genetic disorders, screening for Rhesus factor, screening for aneuploidy and identification of preeclampsia.

Plasma/Serum Cell-Free Circulating DNA Purification Micro Kit Dx

For serum input volumes 10 µL – 200 µL. Purify high-quality DNA in 15-20 minutes

Plasma/Serum Cell-Free Circulating DNA Purification Mini Kit Dx

For serum input volumes 200 µL – 500 µL. Purify high-quality DNA in 15-20 minutes.

Plasma/Serum Cell-Free Circulating DNA Purification Midi Kit Dx

The first column will handle the large volume input of bodily fluids that is followed by a concentration on a mini column for a final elution of 25 µL to 50 µL. All components for the purification & concentration are provided in one convenient & fast kit for the easy processing of large input volumes of bodily fluids. For a schematic workflow of the protocol click here to view. For serum input volumes 1 mL – 4 mL. Purify high-quality DNA in 90 minutes.

Plasma/Serum Cell-Free Circulating DNA Purification Maxi Kit Dx

The first column will handle the large volume input of bodily fluids that is followed by a concentration on a mini column for a final elution of 25 µL to 50 µL. All components for the purification & concentration are provided in one convenient & fast kit for the easy processing of large input volumes of bodily fluids. For a schematic workflow of the protocol click here to view. For serum input volumes 5 mL – 10 mL. Two kits in one – purify and concentrate DNA from bodily fluids using a convenient two column system.

Details

Supporting Data

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Kit Specifications
Minimum Plasma/Serum Input	10 μL
Maximum Plasma/Serum Input	200 μL
Size of DNA Purified	≥ 50 bp
Elution Volume	25-50 μL
Time to Complete 10 Purifications	15-20 minutes
Average Yields*	Variable depending on specimen

*Please check page 7 in the product manual for the Plasma/Serum Average Yields and the Common DNA Quantification Methods.

Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature (15-25°C) for up to 2 years without showing any reduction in performance. Norgen’s Plasma/Serum Cell-Free Circulating DNA Purification Kits contain ready-to-use Proteinase K solution, which is dissolved in a specially prepared storage buffer. The Proteinase K is stable for up to 2.5 years after delivery when stored at room temperature. To prolong the lifetime of Proteinase K, storage at 2–8°C is recommended.

Component	Cat. Dx55100 (50 preps)	Cat. Dx55500 (50 preps)	Cat. Dx55600 (20 preps)	Cat. Dx55800 (10 preps)
Binding Buffer B	2 x 40 mL	40 mL	2 x 85 mL 1 x 12 mL	2 x 85 mL 1 x 40 mL
Proteinase K	2 x 1 mL	0.6 mL	6.5 mL	8 mL
Solution WN	18 mL	18 mL	18 mL	18 mL
Wash Solution A	38 mL	18 mL	18 mL	18 mL
Elution Buffer B	8 mL	8 mL	30 mL	30 mL
Micro Spin Columns	50	50	20	10
Mini Spin Columns	50	–	–	–
Midi Spin Columns	–	–	20	–
Maxi Spin Columns	–	–	–	10
Collection Tubes	100	50	20	10
Elution Tubes (1.7 mL)	100	50	20	10
Product Insert	1	1	1	1