Sample type purification kit guide
The 16S V3-V4 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V3-V4 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 V3-V4 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 V3-V4 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.
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 V3-V4 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. 70400 (24 preps) | Cat. 70410, 70420, 70430, 70440 (96 preps) |
---|---|---|---|
Amplicon PCR (PCR 1) | MGX Master Mix | 330 µL | 1,320 µL |
16S V3-V4 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 |
endo-BCN-OH is a click chemistry handle which readily reacts with azides. The primary alcohol allows it to be ligated to other linkers.
endo-BCN-OH is a click chemistry handle which readily reacts with azides. The primary alcohol allows it to be ligated to other linkers.
HiPure FFPE RNA Kit supplies a simple and rapid RNA extraction for Formalin-fixed, paraffin-embedded (FFPE) tissue and sections samples. This kit is based on silica gel column purification technology, no phenol-chloroform extraction or alcohol precipitation. The whole extraction only takes 30 minutes (not including digestion time). RNA can be directly used for downstream applications such as RT-PCR, Northern blot, vitro translation and other experiments.
Specifications
Features | Specifications |
Main Functions | Isolation total RNA from FFPE tissue and section samples |
Applications | RT-PCR, quantitative RT-PCR, Northern hybridization, Poly A purification, nucleic acid protection and in vitro translation |
Purification method | Mini spin column |
Purification technology | Silica technology |
Process method | Manual (centrifugation or vacuum) |
Sample type | FFPE tissue sample |
Sample amount | 6mg |
Yield | 20μg |
Elution volume | ≥20μl |
Time per run | ≤60 minutes |
Liquid carrying volume per column | 800µl |
Binding yield of column | 100µg |
This product is based on silica column purification. Remove paraffin by Buffer DPS. Sample lysis with proteinase K digestion requires only 15 minutes. After lysis, samples are incubated at 80ºC for 15 minutes. Transfer to an adsorption column and RNA is adsorbed on the membrane, while protein is not adsorbed and is removed with filtration. After washing proteins and other impurities, RNA was finally eluted with low-salt buffer.
Advantages
Kit Contents
Contents | R414302 | D414303 |
Purification Times | 50 Preps | 250 Preps |
HiPure RNA Micro Columns | 50 | 250 |
2ml Collection Tubes | 50 | 250 |
Buffer DPS | 60 ml | 250 ml |
Buffer FRL | 15 ml | 60 ml |
Buffer RLC | 15 ml | 60 ml |
Buffer RWC* | 10 ml | 50 ml |
Buffer RW2* | 20 ml | 2 x 50 ml |
Protease Dissolve Buffer | 1.8 ml | 10 ml |
Proteinase K | 24 mg | 120 mg |
RNase Free Water | 10 ml | 20 ml |
Storage and Stability
Proteinase K should be stored at 2-8°C upon arrival. However, short-term storage (up to 12 weeks at room temperature (15-25°C) does not affect their performance. The remaining kit components can be stored 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 FFPE RNA Kit supplies a simple and rapid RNA extraction for Formalin-fixed, paraffin-embedded (FFPE) tissue and sections samples. This kit is based on silica gel column purification technology, no phenol-chloroform extraction or alcohol precipitation. The whole extraction only takes 30 minutes (not including digestion time). RNA can be directly used for downstream applications such as RT-PCR, Northern blot, vitro translation and other experiments.
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