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 |
Norgen’s EXTRAClean Urine Cell-Free Circulating RNA Purification Kits provide a fast, reliable, reproducible, and simple procedure for isolating circulating RNA and exosomal RNA from various urine inputs ranging from 250 μL and up to 30 mL. The purified RNA is of the highest integrity, and can be used in a number of downstream applications including real-time PCR, reverse transcription PCR, Northern blotting, RNase protection and primer extensions, and expression array assays.
EXTRAClean Cell-Free Circulating RNA Purification Mini Kit:
For sample volumes ranging from 250 μL to 2 mL
EXTRAClean Cell-Free Circulating RNA Purification Midi Kit:
For sample volumes ranging from 2mL to 10mL. The first column will handle the large volume input of urine that is followed by a concentration on a mini column for a final elution of 50 μL to 100 μL
EXTRAClean Cell-Free Circulating RNA Purification Maxi Kit:
For sample volumes ranging from 10 mL to 30 mL. The first column will handle the large volume input of urine that is followed by a concentration on a mini column for a final elution of 50 μL to 100 μL.
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| Kit Specifications | |
|---|---|
| Sample Type | Fresh or frozen urine |
| Sample Volume Range | 10 to 30 mL |
| Minimum Elution Volume | 50 μL |
| Maximum Elution Volume | 100 μL |
| Time to Complete 10 Purifications | 40–50 minutes |
| Size of RNA Purified | All sizes, including miRNA and small RNA (<200 nt) |
| Average Yields ¥ | Variable depending on specimen |
¥Please check page 5 of the protocol for average urine yields and common RNA quantification methods.
All buffers should be kept tightly sealed and stored at room temperature. These kits are stable for 2 years after the date of shipment. It is recommended to warm Lysis Buffer A for 20 minutes at 60°C if any salt precipitation is observed.
| Component | Mini (Cat. 72900) | Midi (Cat. 72910) | Maxi (Cat. 72920) |
|---|---|---|---|
| Binding Solution K | 25 mL | 75 mL | 1 x 75 mL 1 x 25 mL |
| Lysis Buffer A | 30 mL | 20 mL | 20 mL |
| Wash Solution A | 18 mL | 18 mL | 18 mL |
| Elution Solution A | 6 mL | 6 mL | 6 mL |
| Collection Tubes | 50 | 20 | 10 |
| Elution Tubes (1.7 mL) | 50 | 20 | 10 |
| EXTRAClean Mini Spin Columns | 50 | 20 | 10 |
| EXTRAClean Midi Spin Columns | – | 20 | – |
| EXTRAClean Maxi Spin Columns | – | – | 10 |
| Product Insert | 1 | 1 | 1 |
DBCO-PEG5-NHS Ester is a DBCO-containing PEG linker containing NHS ester that can react specifically and efficiently with primary amines (e.g. the side chain of lysine residues or aminosilane-coated surfaces) at neutral or slightly basic condition to form a covalent bond. PEG5 spacer increases the water solubility of compounds in aqueous media. DBCO is commonly used for copper-free Click Chemistry reaction. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.
DBCO-PEG5-NHS Ester is a DBCO-containing PEG linker containing NHS ester that can react specifically and efficiently with primary amines (e.g. the side chain of lysine residues or aminosilane-coated surfaces) at neutral or slightly basic condition to form a covalent bond. PEG5 spacer increases the water solubility of compounds in aqueous media. DBCO is commonly used for copper-free Click Chemistry reaction. Reagent grade, for research purpose. Please contact us for GMP-grade inquiries.