This kit enables the rapid purification of amplified DNA products from PCR mixes. It is able to effectively remove PCR by-products including primers, dimers, enzymes, unincorporated nucleotides and mineral oil from the desired PCR product. The purified PCR products are fully compatible with restriction enzyme digestion, ligation into vectors, labeling, sequencing and more. This kit can also be used as an alternative to organic extraction and ethanol precipitation to clean up various enzymatic reactions.
The kit is also available in a 96-well format for high-throughput PCR purification. Purification with the 96-well plate can be performed using either a vacuum manifold or centrifugation.
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| Kit Specifications – Spin Column | |
| Column Binding Capacity | 10 μg |
| Size of DNA Purified | 100 – 15,000 bp |
| Average Recovery | > 90% |
| Average Primer Removal | > 90% |
| Minimum Elution Volume | 30 μL |
| Time to Complete 10 Purifications | 15 minutes |
Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 1 year after the date of shipment.
| Component | Cat. 14400 (50 preps) | Cat. 45700 (250 preps) | Cat. 24800 (192 preps) |
|---|---|---|---|
| Binding Buffer C | 30 mL | 5 x 30 mL | 3 x 30 mL |
| Wash Solution A | 12 mL | 2 x 20 mL | 2 x 38 mL |
| Elution Buffer B | 8 mL | 2 x 30 mL | 2 x 15 mL |
| Spin Columns | 50 | 250 | – |
| Collection Tubes | 50 | 250 | – |
| 96-Well Plate | – | – | 2 |
| Adhesive Tape | – | – | 4 |
| 96-Well Collection Plate | – | – | 2 |
| Elution Tubes (1.7 mL) | 50 | 250 | – |
| 96-Well Elution Plate | – | – | 2 |
| Product Insert | 1 | 1 | 2 |
Sample type purification kit guide
The 16S V3-V5 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-V5 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.
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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 V3-V5Library 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. 70500 (24 preps) | Cat. 70510, 70520, 70530, 70540 (96 preps) |
|---|---|---|---|
| Amplicon PCR (PCR 1) | MGX Master Mix | 330 µL | 1,320 µL |
| 16S V3-V5 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 |
With the development of molecular biology, stool, a new non-invasive sample, has been widely used in the research of animal molecular genetics, population ecology, behavioral ecology and some intestinal disease diagnosis. Stool samples includes gut microbial DNA, food residue sample DNA, and alimentary tract exfoliated cell DNA.
The primary problem encountered when using stool sample for molecular biology research is the low content of exfoliated cells in the digestive tract and a certain degree of degradation of genetic material in stool. Another issue in molecular scatology research based on PCR is the presence of a large number of inhibitors in stool that can affect Taq enzyme activity, leading to downstream detection inactivation. These inhibitors include polysaccharides, plant polysaccharides, bile acids, bile salts, bile pigments, digestive juices, mucus, etc. Therefore, selecting appropriate extraction methods to obtain high-quality DNA is the key to successful downstream detection of stool DNA.
At present, the pretreatment methods used in the laboratory, such as phenol/chloroform extraction, cetyltrimethyl bromide (CTAB) lysis, and guanidine isothiocyanate lysis, lack universality in different species, and the success rate of extracting DNA for PCR amplification is also very low. The HiPure Stool DNA Kit provided by Magen Company has opened up a new approach for DNA extraction from stool samples with good universality, high cost-effectiveness, high yield and purification. The reagent kit adopts a unique solution system and inhibitory factor adsorbent, which can efficiently remove various impurities in stool samples. The purified DNA can be directly used for PCR, quantitative PCR and other applications.
This product allows rapid and reliable isolation of high-quality genomic DNA from various stool samples. Up to 100 mg soil samples can be processed in 60 minute. The system combines the reversible nucleic acid binding properties of HiPure matrix with the speed and versatility of spin column technology to eliminate PCR inhibiting compounds such as humic acid from soil samples. Purified DNA is suitable for PCR, restriction digestion, and next-generation sequencing. There are no organic extractions thus reducing plastic waste and hands-on time to allow multiple samples to be processed in parallel.
Specifications
| Features | Specifications |
| Main Functions | Isolation total DNA from 50-100mg stool samples |
| Applications | PCR, Southern Blot, enzyme digestion and NGS, etc. |
| Purification method | Mini spin column |
| Purification technology | Silica technology |
| Process method | Manual (centrifugation or vacuum) |
| Sample type | Stool |
| Sample amount | 50-100mg |
| Yield | 3-15μg |
| Elution volume | ≥30μl |
| Time per run | ≤60 minutes |
| Liquid carrying volume per column | 750μl |
| Binding yield of column | 100μg |
Stool sample is homogenized and then treated in a specially formulated buffer containing detergent to lyse bacteria, yeast, and fungal samples. Humic acid, proteins, polysaccharides, and other contaminants are removed using our proprietary Absorber Solution. Binding conditions are then adjusted and the sample is applied to a DNA Mini Column. Two rapid wash steps remove trace contaminants and pure DNA is eluted in low ionic strength buffer. Purified DNA can be directly used in downstream applications without the need for further purification.
Kit Contents
| Contents | D314102 | D314103 |
| Purification Times | 50 Preps | 250 Preps |
| HiPure DNA Mini Columns II | 50 | 250 |
| 2ml Collection Tubes | 50 | 250 |
| 2ml Bead Tubes | 50 | 250 |
| Proteinase K | 24 mg | 120 mg |
| Protease Dissolve Buffer | 1.8 ml | 10 ml |
| Buffer SPL | 40 ml | 200 ml |
| Buffer PCI | 40 ml | 200 ml |
| Buffer AL | 20 ml | 80 ml |
| Buffer GW1 | 22 ml | 88 ml |
| Buffer GW2 | 20 ml | 2 x 50 ml |
| Buffer AE | 15 ml | 30 ml |
Storage and Stability
Proteinase K and Buffer PCI 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.
With the development of molecular biology, stool, a new non-invasive sample, has been widely used in the research of animal molecular genetics, population ecology, behavioral ecology and some intestinal disease diagnosis. Stool samples includes gut microbial DNA, food residue sample DNA, and alimentary tract exfoliated cell DNA.
The primary problem encountered when using stool sample for molecular biology research is the low content of exfoliated cells in the digestive tract and a certain degree of degradation of genetic material in stool. Another issue in molecular scatology research based on PCR is the presence of a large number of inhibitors in stool that can affect Taq enzyme activity, leading to downstream detection inactivation. These inhibitors include polysaccharides, plant polysaccharides, bile acids, bile salts, bile pigments, digestive juices, mucus, etc. Therefore, selecting appropriate extraction methods to obtain high-quality DNA is the key to successful downstream detection of stool DNA.
At present, the pretreatment methods used in the laboratory, such as phenol/chloroform extraction, cetyltrimethyl bromide (CTAB) lysis, and guanidine isothiocyanate lysis, lack universality in different species, and the success rate of extracting DNA for PCR amplification is also very low. The HiPure Stool DNA Kit provided by Magen Company has opened up a new approach for DNA extraction from stool samples with good universality, high cost-effectiveness, high yield and purification. The reagent kit adopts a unique solution system and inhibitory factor adsorbent, which can efficiently remove various impurities in stool samples. The purified DNA can be directly used for PCR, quantitative PCR and other applications.
This product allows rapid and reliable isolation of high-quality genomic DNA from various stool samples. Up to 100 mg soil samples can be processed in 60 minute. The system combines the reversible nucleic acid binding properties of HiPure matrix with the speed and versatility of spin column technology to eliminate PCR inhibiting compounds such as humic acid from soil samples. Purified DNA is suitable for PCR, restriction digestion, and next-generation sequencing. There are no organic extractions thus reducing plastic waste and hands-on time to allow multiple samples to be processed in parallel.