Introduction

Soil samples contain a large number of microorganisms, the vast majority of which can not be directly cultivated for reproduction and research. Extracting DNA from soil samples is the most effective method for studying soil microorganisms. At present, there are mainly direct and indirect methods for extracting microbial DNA from soil samples. The direct method refers to placing soil samples in the lysis solution, and using effective wall breaking methods to release all microbial DNA into the lysis solution, followed by separation and extraction, such as Zhou’s method. Indirect method refers to placing soil in a buffer, such as Buffer PBS, to separate microorganisms from the soil and then extract DNA. The indirect method can greatly reduce the impact of humic acids and heavy metal salts on DNA extraction in soil, but this method will lose many microorganisms and the resulting DNA is not the entire genome (metagenome) of the soil sample. Currently, few researchers have adopted this method. Extracting DNA directly from soil samples can maximize the likelihood of obtaining the entire genome, but this method faces the following issues:

1. Humic acid pollution. The soil, especially in forests and grasslands, is rich in humic acids. Humic acid is a series of organic molecules, some of which are very similar to nucleic acid molecules and difficult to remove during purification. Trace amounts of humic acid pollution can lead to downstream applications such as PCR and enzyme digestion failure.

2. Lysis method. Soil samples contain various microorganisms, such as bacteria and fungi. Gram positive bacteria and fungi both contain very thick bacterial walls, and effectively breaking down the cell walls of these microorganisms is crucial for extracting high-yield metagenomic DNA. Due to the complexity of soil samples, it is not feasible to use enzymatic methods (such as lysozyme, wall breaking enzyme, snail enzyme) or liquid nitrogen grinding, as the soil contains various metalions or inhibitory factors that inactive the digestive enzymes, or the presence of sand particles in the soil makes liquid nitrogen grinding difficult.

3. The DNA yield is difficult to control. Soil samples would have significant changes in the number and variety of microorganisms due to fertility, inferiority, high moisture content, dryness, or depth of sampling. In a small range of soil samples, the DNA content often varies by thousands of times. In addition, certain chemical components in soil, such as heavy metal salts and clay substances, can cause a decrease in DNA yield.

Magen’s HiPure Soil DNA Kits are currently the most optimized kit for soil DNA extraction. The kit adopts glass bead grinding method and thermal shock chemical wall breaking method, which can be carried out in the point vortex instrument without special bead grinding instrument, and is suitable for a wide range of laboratories. The Absorber Solution in the reagent kit is a humic acid adsorbent exclusively developed by Magen Company, which can efficiently remove various humic acid pollutants. In addition, an alcohol-free silica gel column purification method is also used to efficiently remove various soluble metal salts and other soluble inhibitory factors from the soil. The kit has successfully extracted from the following soil (partially based on customer feedback): soil from forests in nature reserves (30 to 40 years old forest soil with a surface layer of 30-50cm deciduous layer), mangrove soil, grasslands, farmland, seabed mud, sludge, mineral area soil, organic matter contaminated soil, pond mud, garbage mud, air conditioning pipeline deposits, etc.

This product allows rapid and reliable isolation of high-quality genomic DNA from various soil samples. Up to 500 mg soil samples can be processed in 60 minute. The system combines the reversible nucleic acid binding properties of HiPure matrix with the speed and versatilityof spin column technology to eliminate PCR inhibiting compounds such as humic acid from soil samples. Purified DNA is suitable for PCR, restriction digestion, and next-generation sequencing. There are no organic extractions thus reducing plastic waste and hands-on time to allow multiple samples to be processed in parallel.

Details

Specifications

Features	Specifications
Main Functions	Isolation DNA from 200-500mg soil sample
Applications	PCR, southern blot and enzyme digestion, etc.
Purification method	Mini spin column
Purification technology	Silica technology
Process method	Manual (centrifugation or vacuum)
Sample type	Soil
Sample amount	200-500mg
Elution volume	≥30μl
Time per run	≤60 minutes
Liquid carrying volume per column	800μl
Binding yield of column	100μg

Principle

Soil sample is homogenized and then treated in a specially formulated buffer containing detergent to lyse bacteria, yeast, and fungal samples. humic acid,proteins, polysaccharides, and other contaminants are removed using our proprietary Absorber Solution. Binding conditions are then adjusted and the sample is applied to a DNA Mini Column. Two rapid wash steps remove trace contaminants and pure DNA is eluted in low ionic strength buffer. Purified DNA can be directly used in downstream applications without the need for further purification.

Advantages

• Fast – several samples can be extracted in 40 minutes (after digestion)
• High purity – purified DNA can be directly used in various downstream applications
• Good repeatability – silica technology can obtain ideal results every time
• High recovery – DNA can be recovered at the level of PG

Kit Contents

Contents	D314202	D314203
Purification Times	50 Preps	250 Preps
Hipure DNA Mini Columns II	50	250
2ml Collection Tubes	50	250
2ml Bead Tubes	50	250
Buffer SOL	60 ml	250 ml
Buffer SDS	5 ml	20 ml
Buffer PS	10 ml	50 ml
Absorber Solution	10 ml	50 ml
Buffer GWP	40 ml	220  ml
Buffer DW1	30 ml	150  ml
Buffer GW2*	20 ml	2 x 50  ml
Buffer AE	15 ml	30 ml

Storage and Stability

Absorber Solution should be stored at 2-8°C upon arrival. However, short-term storage (up to 24 weeks) at room temperature (15-25°C) does not affect their performance. The remaining kit components can be stored dry at room temperature (15-25°C) and are stable for at least 18 months under these conditions.

Experiment Data

Soil samples contain a large number of microorganisms, the vast majority of which can not be directly cultivated for reproduction and research. Extracting DNA from soil samples is the most effective method for studying soil microorganisms. At present, there are mainly direct and indirect methods for extracting microbial DNA from soil samples. The direct method refers to placing soil samples in the lysis solution, and using effective wall breaking methods to release all microbial DNA into the lysis solution, followed by separation and extraction, such as Zhou’s method. Indirect method refers to placing soil in a buffer, such as Buffer PBS, to separate microorganisms from the soil and then extract DNA. The indirect method can greatly reduce the impact of humic acids and heavy metal salts on DNA extraction in soil, but this method will lose many microorganisms and the resulting DNA is not the entire genome (metagenome) of the soil sample. Currently, few researchers have adopted this method. Extracting DNA directly from soil samples can maximize the likelihood of obtaining the entire genome, but this method faces the following issues:

Gel images of different ranges of library size selection. Sheared human genomic DNA was used as input.

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Library size selection is an enrichment of a specific range of library sizes for NGS library preparations. The NGS library preparation is related to the quality of the sequencing data. Precise NGS library size selection can increase sequencing efficiency, improve data quality, and reduce costs.

There are two types of sequencing technologies: short-read sequencing and long-read sequencing. Short-read sequencing uses DNA libraries that contain small insert DNA fragments of similar sizes, usually several hundred base pairs. The sequencing efficiency can be improved if the DNA size selection is in the right range. Cat.# 20104S and 20104L are the best kits for NGS library size selection of illumina paired-end 100 (PE100) sequencing with 100-200 bp library inserts; Cat.# 20105S and 20105L are the best kits for NGS library size selection of illumina paired-end 150 (PE150) sequencing with 150-300 bp library inserts; and Cat.# 20106S and 20106L are the best kits for NGS library size selection of illumina paired-end 300 (PE300) sequencing with 300-600 bp library inserts.

Long-read sequencing uses a large DNA fragment as input and makes very long reads. Usually, library size selection is preferred to remove smaller fragments. Cat.# 20110S and 20110L are the best kits for long-read sequencing size selection with DNA sizes >5 kb, and Cat.# 20111S and 20111L are the best kits for long-read sequencing size selection with DNA sizes >10 kb.

The magnetic beads, or SPRI (Solid Phase Reversible Immobilization) beads, is well used for the purification of DNA due to their reversible DNA binding. The NGS library can be size-selected by the magnetic beads or SPRI beads. The properties of the magnetic beads can be changed for a specific range of DNA binding. The contaminants and other unwanted components in the libraries can also be removed during size selection.

Specific ranges of NGS libraries can be selected using magnetic beads with different buffer compositions. The first DNA-beads binding step, also called the right-side clean-up, removes large NGS library fragments. The large NGS library fragments that bind to the beads are discarded with the beads pellet. The desired NGS library fragments in the supernatant are transferred to a new well, and new beads are added to the supernatant for the second beads-DNA binding, also called the left-side clean-up. After the rinsing step, the NGS library fragments with the dual selection are eluted in water or an appropriate buffer. The magnetic beads method has great advantages over time-consuming column purification and tedious gel-based purification.

NGS library size selection with dual clean-ups.

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Library size selection for long-read sequencing only requires a single clean-up. In this case, only the large library fragments are bound to the beads, while other small library fragments are discarded with the supernatant. The selected larger library fragments are eluted in water or an appropriate buffer after the rinsing step.

NGS library size selection with single clean-up for >5 kb and >10 kb libraries.

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Features of NGS library size selection

• • • 5 ranges for NGS library size selection
      • • illumina PE100 sequencing: 250-350 bp
        • illumina PE150 sequencing: 300-450 bp
        • illumina PE100 sequencing: 450-750 bp
        • Long-read sequencing: >5 kb
        • Long-read sequencing: >10 kb
    • Rapid protocol: only 20 minutes
    • Simple workflow
      • • No columns required
        • No gel purification required
        • No centrifugation required

Overview

• Protocol optimized for DNA isolated from a diversity of samples including stool, soil, water, saliva, plant, urine, skin, and more
• Simple and quick workflow: library could be prepared in less than 5 hours
• Component of Norgen’s metagenomics workflow
• A single NGS run can be prepared with up to 384 unique dual-index libraries

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.

Details

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