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Sample type purification kit guide
The 16S V1-V3 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V1-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 V1-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 V1-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.
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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-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. 70200 (24 preps) | Cat. 70210, 70220, 70230, 70240 (96 preps) |
|---|---|---|---|
| Amplicon PCR (PCR 1) | MGX Master Mix | 330 µL | 1,320 µL |
| 16S V1-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 |
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.
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 |
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
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.