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 |
N-(t-butyl ester-PEG4)-N-bis(PEG4-Propargyl) is a trifunctional PEG linker that combines a t-butyl ester with two terminal alkynes. The alkynes can be applied in copper-click chemistry with azides to form stable triazole linkages with a target molecule, while the t-butyl ester can be deprotected and reacted with amines or alcohols to form amides or esters.
N-(t-butyl ester-PEG4)-N-bis(PEG4-Propargyl) is a trifunctional PEG linker that combines a t-butyl ester with two terminal alkynes. The alkynes can be applied in copper-click chemistry with azides to form stable triazole linkages with a target molecule, while the t-butyl ester can be deprotected and reacted with amines or alcohols to form amides or esters.
HiPure DNA Clean Up 96 Kit uses proprietary chemistry and HiPure technology to recover DNA Fragments between 60bp-10kbp with yields exceeding 80%. DNA is suitable for ligations, PCR, sequencing, restriction digestion, or various labeling reactions. In addition, this kit can be also used to recover DNA directly from enzymatic reactions such as PCR and enzyme digestion reactions.
Specifications
Features | Specifications |
Main Functions | Recover DNA fragments between 60bp-10kbp from PCR, Reactions, crude DNA using 96 well Bind Plate |
Applications | PCR, sequencing, labeling reactions, ligations and restriction digestion, etc. |
Purification method | 96 well Bind Plate |
Purification technology | Silica technology |
Process method | Manual (centrifugation or vacuum) |
Sample type | PCR product, enzymatic reaction |
Sample amount | Appropriate |
Recovery | ≥80% |
Elution volume | ≥75μl |
Time per run | ≤60 minutes |
Liquid carrying volume per column | 800µl |
Binding yield of column | 20µg |
The HiPure system uses a simple bind-wash-elute procedure. Gel slices are dissolved in a buffer containing a pH indicator, allowing easy determination of the optimal pH for DNA binding, and the mixture is applied to the column. Nucleic acids adsorb to the silica-gel membrane in the high-salt conditions provided by the buffer. Impurities are washed away and pure DNA is eluted with a small volume of low-salt buffer provided or water, ready to use in subsequent applications.
Advantages
Kit Contents
Contents | D212201 | D212202 | D212203 |
Purification Times | 1 x 96 Preps | 4 x 96 Preps | 20 x 96 Preps |
Buffer GDP | 120 ml | 400 ml | 4 x 400 ml |
Buffer DW2 | 20 ml | 100 ml | 3 x 100 ml |
Elution Buffer | 20 ml | 20 ml | 30 ml |
HiPure DNA Plate | 1 | 4 | 20 |
2.2ml Collection Plate | 1 | 4 | 20 |
1.6 ml Collection Plate | 1 | 4 | 20 |
0.8 ml Collection Plate | 1 | 4 | 20 |
Storage and Stability
The Kit can be stored dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions. If any precipitates form in the buffers, warm at 37℃ to dissolve.
HiPure DNA Clean Up 96 Kit uses proprietary chemistry and HiPure technology to recover DNA Fragments between 60bp-10kbp with yields exceeding 80%. DNA is suitable for ligations, PCR, sequencing, restriction digestion, or various labeling reactions. In addition, this kit can be also used to recover DNA directly from enzymatic reactions such as PCR and enzyme digestion reactions.
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