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 |
Usages:
For isolating lactose-fermenting Gram-negative enteric bacilli.
Principle:
Peptones are sources of nitrogen and other nutrients. Lactose is a fermentable carbohydrate. When lactose is fermented, alocal pH drop around the colony causes a color change in the pH indicator (neutral red) and bile precipitation. Bile salts,bile salts no. 3, oxgall and crystal violet are selective agents that inhibit growth of gram-positive organisms. Agar is the solidifying agent.
Formulation（per liter）:
| Pancreatic Digest of Gelatin | 17.0 g |
| Peptones (meat and casein) | 3.0 g |
| Lactose Monohydrate | 10.0 g |
| Sodium Chloride | 5.0 g |
| Bile Salts | 1.5 g |
| Agar | 13.5 g |
| Neutral Red | 30.0 mg |
| Crystal Violet | 1 mg |
| Final pH | 7.1±0.2 |
How to use：
1.Suspend 50 g in 1 L of distilled or deionized water. Heat to boiling to dissolve completely. Autoclave at 121°C for 15 minutes.
2.Transfer 1 mL of Soybean–Casein Digest Broth to 100 mL of MacConkey Broth, and incubate at 42 to 44 for 24 to 48 hours. Subculture on a plate of MacConkey Agar at 30 to 35 deg.C for 18 to 72 hours.
Quality control：
| Item | The name and number of strain | PR/G | Reaction |
| Growth rate | E.Coli ATCC8739 | PR≥0.7 | Rose-red |
| Characteristic difference | Proteus mirabilis CMCC(b)49005 | PR≥0.7 | Colorless, no swarming |
| Selective | Staphylococcus aureus ATCC6538 | G≤1 | － |
Storage: Keep container tightly closed, store in a cool, dry place, away from bright light. Storage period of three years.
Specifications: 250g/bottle
250g
Mastitis is the single most costly disease of dairy cattle resulting in the reduction of milk yield and quality. The inflammation of the udder is mainly caused by infection of various bacteria. One of such mastitis bacteria, Streptococcus agalactiae, is highly infectious and causes mainly subclinical infections, which are not identified by the herdsman. As a result, S. agalactiae can spread widely within a herd, causing immediate loss due to reduced milk. S. agalactiae is a gram-positive bacteria belonging to the Group B streptococci. Traditional cultural identification of S. agalactiae is based on S. agalactiae being beta-hemolytic as well as presence of group B Lancefield antigen and by its ability to hydrolyze sodium hippurate.
Streptococcus agalactiae TaqMan PCR Kit, 100 reactions
Streptococcus agalactiae TaqMan PCR Probe/Primer Set and Controls, 100 reactions
For research use only and NOT intended for in vitro diagnostics.
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Storage Conditions and Product Stability
All kit components can be stored for 2 years after the date of production without showing any reduction in performance.
All kit components should be stored at -20°C upon arrival. Repeated thawing and freezing (> 2 x) of the Master Mix and Positive Control should be avoided, as this may affect the performance of the assay. If the reagents are to be used only intermittently, they should be frozen in aliquots.
| Component | Cat. TM30650 (100 preps) | Cat. TM30610 (100 preps) |
|---|---|---|
| MDx TaqMan 2X PCR Master Mix | 2 x 700 μL | – |
| S. agalactiae Primer & Probe Mix | 280 μL | 280 μL |
| S. agalactiae Positive Control | 150 μL | 150 μL |
| Nuclease-Free Water (Negative Control) | 1.25 mL | 1.25 mL |
| Product Insert | 1 | 1 |