Sample type purification kit guide
The 16S V1-V2 Library Preparation Kit for Illumina consists of the reagents and components required for library preparation of the 16S V1-V2 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-V2 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-V2 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-V2 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. 70100 (24 preps) | Cat. 70110, 70120, 70130, 70140 (96 preps) |
|---|---|---|---|
| Amplicon PCR (PCR 1) | MGX Master Mix | 330 µL | 1,320 µL |
| 16S V1-V2 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 |
The HighRanger 1 kb DNA Ladder is prepared to ensure quality and batch-to-batch consistency. This Ladder contains thirteen discrete fragments ranging from 300 bp to 10,000 bp with a higher intensity reference band at 5000 bp. This Ladder is ideal for quick sizing of PCR products and restriction digests.
Contents
1mL of premixed DNA ladder (0.5µg/10µL) in loading buffer (10mM EDTA, 10% glycerol, 0.015% bromophenol blue, and 0.17% SDS).
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HighRanger 1kb DNA Ladder (Cat# 11900) – 100 loads
Ladder Properties:
• Thirteen discrete bands, ranging from 300 bp to 10000 bp
• Higher intensity band at 5000 bp for easy reference
| Fragment | Size (bp) | Mass (ng) |
| 1 | 10000 | 58 |
| 2 | 8000 | 53 |
| 3 | 6000 | 46 |
| 4 | 5000 | 67 |
| 5 | 4000 | 56 |
| 6 | 3000 | 47 |
| 7 | 2500 | 41 |
| 8 | 2000 | 34 |
| 9 | 1500 | 29 |
| 10 | 1000 | 18 |
| 11 | 700 | 13 |
| 12 | 500 | 23 |
| 13 | 300 | 14 |
Recommended Use:
Mix thoroughly. For best results, load 10µL of DNA ladder per well. For precise mass determination with a densitometer, stain gel after electrophoresis using 0.5µg/mL ethidium bromide for 30-40 minutes. The table above shows the size and mass for each band based on 10µL ladder per well.
Storage:
This kit is stable for 2 years after the date of shipment.
This ladder was standardized using 10µL of DNA per lane on a 0.8 cm thick, 13 x 15 cm, 1.0% agarose gel run in TAE buffer.
