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 |
Norgen’s Plasma/Serum Circulating DNA Purification Kits (Slurry Format) provide efficient methods for the purification of fragmented free-circulating DNA from human plasma or serum. The kit is able to isolate all sizes of circulating DNA. The slurry format provides an advantage over other available kits in that it does not require extension tubes for the purification of free-circulating DNA from large sample volumes. DNA can be isolated from either fresh or frozen samples using this kit. The kit will also isolate viral and bacterial DNA from plasma/serum. Typical yields of purified free-circulating DNA will vary depending on the input sample (1-100ng/mL circulating DNA in human plasma), with more concentrated samples tending to yield more free-circulating nucleic acids. The purified, inhibitor-free plasma/serum free-circulating DNA is eluted in an elution solution that is compatible with PCR, qPCR, methylation-sensitive PCR and Southern Blot analysis.
Plasma/Serum Circulating DNA Purification Mini Kit (Slurry Format)
Norgen’s Plasma/Serum Circulating DNA Purification Mini Kit (Slurry Format) provides a fast, reliable and simple procedure for isolating circulating DNA from various amounts of plasma/serum ranging from 50 μL to 400 μL. Preparation time for a single sample is less than 30 minutes.
Plasma/Serum Circulating DNA Purification Midi Kit (Slurry Format)
Norgen’s Plasma/Serum Circulating DNA Purification Midi Kit (Slurry Format) provides a fast, reliable and simple procedure for isolating circulating DNA from various amounts of plasma/serum ranging from 400 μL to 2 mL. Preparation time for a single sample is less than 45 minutes.
Plasma/Serum Circulating DNA Purification Maxi Kit (Slurry Format)
Norgen’s Plasma/Serum Circulating DNA Purification Maxi Kit (Slurry Format) provides a fast, reliable and simple procedure for isolating circulating DNA from various amounts of plasma/serum ranging from 2 mL to 10 mL. Preparation time for a single sample is less than 45 minutes.
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| Kit Specifications | |
| Minimum Plasma/Serum Input | 50 μL |
| Maximum Plasma/Serum Input | 400 μL |
| Minimum Elution Volume | 100 μL |
| Time to Complete Purifications | < 30 minutes |
Storage Conditions and Product Stability
All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.
| Component | Cat. 50600 (50 preps) | Cat. 51200 (20 preps) | Cat. 51300 (10 preps) |
|---|---|---|---|
| Lysis Buffer D | 65 mL | 125 mL | 3 x 125 mL |
| Slurry A1 | – | 6 mL | – |
| Slurry A2 | 12 mL | – | 12 mL |
| Binding Buffer B | 20 mL | 6 mL | 20 mL |
| Binding Buffer C | – | – | 30 mL |
| Wash Solution A | 2 x 38 mL | 2 x 38 mL | 3 x 38 mL |
| Elution Buffer B | 8 mL | 8 mL | 15 mL |
| Mini Filter Spin Columns | 50 | 20 | – |
| Midi Filter Spin Columns | – | – | 10 |
| Midi Collection and Elution Tubes | – | – | 20 |
| Collection Tubes | 50 | 40 | 10 |
| Spin Columns | – | 20 | 10 |
| Elution Tubes (1.7 mL) | 50 | 40 | 10 |
| Product Insert | 1 | 1 | 1 |
Collagen is a fundamental component of the extracellular matrix, and the predominant protein in animals, constituting around 30% of total protein mass. A glycoprotein, it is well known for its triple helical structure. This is formed from three polypeptide α-chains with Gly-X-Y repeating residues (Gly for Glycine, X for proline, and Y for hydroxyproline).
Over 28 types of collagens have been identified, with Type I collagen being the most abundant. It’s prevalent in ligaments, tendons, skin, and bone tissue. Its mature, insoluble form grants it remarkable strength, making it vital for the mobility of organisms. Collagen also has biochemical functions, influencing cell growth, proliferation, and differentiation.
This version of the kit is designed to detect and measure SOLUBLE forms of collagen. Chose the Sircol Insoluble collagen kit if you need to analyse INSOLUBLE collagen.
Collagen, with its diverse properties, finds utility in various industries. It plays a role in medicine for wound healing and has an expanding role in tissue engineering and cell culture for biomedical purposes. It’s gaining popularity in the cosmetic industry for skin rejuvenation and is used in chemical formulations and the food industry as a functional food supplement and additive.
The Sircol 2.0 dye reagent includes Sirius Red, a linear anionic dye with sulfonic acid side chains. This reagent is specially formulated to bind to the Gly-X-Yn helical structure of soluble collagen under assay conditions.
*The improved formulation of Sircol 2.0 dye enables a greater degree of dye-collagen specificity (compared to our previous S1000 assay kit).
Step 1. Prepared samples are placed in the wells of the assay microplate, together with Sircol Dye Reagent. After 30 minutes mixing, any collagen-dye complexes will form as a precipitate. These are collected on the base of the microplate wells by centrifugation.
Step 2. Unbound dye is removed by gentle aspiration, followed by a rinse with Plate Wash Reagent.
Step 3. Following further centrifugation, collagen-bound dye is eluted by incubation with a Dye Release Reagent. Eluted dye is detected ‘in-situ’ by spectrophotometric analysis of the microplate at 556nm.
Step 4. The collagen content of unknown samples can be quantified by comparison against a calibration curve, prepared using the Collagen Reference Standard supplied with the kit.
A list of suggested sample types can be found under the ‘Assay Specification‘ tab.
2 – 200µg/ml or 0.25 – 20µg/ml (using optional Collagen Concentration Protocol)
2 µg/ml
Colorimetric Detection (556nm) (Endpoint), Requires a microplate centrifuge.
96 in total (allows a maximum of 41 samples to be run in duplicate alongside a standard curve).
Soluble* collagens from mammalian**:
In-vivo: Tissues, cartilages and fluids.
In-vitro: Extracellular matrices / Conditioned media from 2D/3D culture environments.
The straightforward sample processing and analysis of Sirco 2.0 make it a good alternative to conventional hydroxyproline analysis.
*Prior salt/acid/acid-pepsin extraction may be necessary to release soluble collagen.
**Sircol 2.0 is primarily designed for use with in-vivo / in-vitro samples of mammalian origin. Collagens originating from other taxonomic groups and kingdoms can also be analysed. See note on p6 of manual for further information.
This kit is designed for research use only. Not for use in diagnostic procedures.
Kit requires access to a microplate centrifuge* (see note below), as well as a spectrophotometer/colorimeter capable of absorbance detection at 556nm.
Specific sample preparation protocols may require customer to provide further reagents, consult assay manual for further information.
*As a minimum, we recommend that the centrifuge can centrifuge a 96-well microplate at 400 x g for 120 minutes. Higher speed centrifuges are recommended (up to a maximum of 2000 x g), allowing a reduction in centrifuge time.
1. Dye Reagent (1x20ml)
2. Collagen Reference Standard (1x5ml, 200µg/ml of soluble Bovine collagen)
3. Plate Wash Reagent (1x28ml)
4. Collagen Concentration Reagent (1x25ml)
5. Neutralisation Reagent (1x8ml)
6. Dye Release Reagent (1x25ml)
7. Assay Microplate (1×96-wells)
8. Microplate Seals (6x)
9. Documentation (QuickStart Guide / Manual / Certificate of Analysis)
NB: Additional reagents may be required for sample preparation prior to assay. Consult manual or contact us for further details. This kit requires the use of a microplate centrifuge, capable of centrifuging a 96-well microplate at 400 x g for 120 minutes. Higher speed centrifuges are recommended (up to a maximum of 2000 x g), allowing a reduction in centrifuge time.
Experience user-friendly detection & measurement of Soluble Collagen with Sircol™ 2.0! Our latest kit simplifies collagen quantification within in-vivo / in-vitro samples. Sircol 2.0 offers enhanced sensitivity and accuracy compared to our previous Sircol kit.