Extraction method for soil microorganism metagenome DNA and corresponding kit

A soil microorganism and metagenome technology, applied in the field of biological genomic DNA extraction, can solve problems such as low extraction efficiency and inability to meet sequencing requirements, and achieve the effects of improving extraction rate, strong practicability and good purity

Inactive Publication Date: 2017-12-01
广东美格基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to: overcome the problems of low extraction efficiency and inability to meet the sequencing requirements in the existing conventional extreme environment soil microbial DNA extraction, and provid

Method used

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  • Extraction method for soil microorganism metagenome DNA and corresponding kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Reagent configuration

[0037] (1) Buffer A: 0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M NaH 2 PO 4 and 0.1MNa 2 HPO 4 , the pH was adjusted to 8.0, the volume was adjusted to 1L with deionized water, and sterilized under high temperature and high pressure.

[0038] (2) 50 mg / ml lysozyme: 20 mM Tris-HCl, 2 mM EDTA, Triton x-100 with a volume fraction of 1.2%.

[0039] (3) 10wt% CTAB: weigh 1g of CTAB powder, and dilute to 100ml with sterile water (requires heating in a 65°C water bath to dissolve).

[0040] (4) Buffer B: Weigh 0.5g of CTAB, and dilute to 50ml with buffer A (heating in a water bath at 65°C is required to dissolve).

[0041] (5) 20% SDS: Weigh 10 g of SDS, and dilute to 50 ml with sterile water (heating in a water bath at 68° C. to dissolve, and adjust the pH to 7.2).

[0042] (6) Phenol: chloroform: isoamyl alcohol (25:24:1).

[0043] (7) Chloroform: isoamyl alcohol (24:1).

[0044] (8) Precooling with isopropanol (4°C), 70% ethanol.

[0045] (...

Embodiment 2

[0067] Example 2 Using the SDS method to extract the sandy soil metagenome

[0068] (1) Take 15g of sand in a 50ml centrifuge tube, add 30ml of buffer A, vortex and mix, and bathe in 55°C water for 20min;

[0069] (2) 9000g, centrifuge for 10min, discard the supernatant;

[0070] (3) Add 10ml of buffer A to the centrifuge tube, and use the freeze-thaw method to disrupt the cells: -80°C for 15 minutes—65°C for 5 minutes (repeat 3 times, and do not need to invert and mix during the 65°C warm bath).

[0071] (4) Cool at room temperature (~10min is enough), add 1ml lysozyme (10mg / ml) to the centrifuge tube (final concentration is about 0.5mg / ml), and bathe in 37℃ water for 1h (during this period, gently invert and mix well every 10min) ).

[0072] (5) Add 0.5ml 10% SDS (final concentration is about 0.5% SDS) and 20μl proteinase K (final concentration is about 100μg / ml), bathe in 58℃ water for 1h (mix up and down every 10min, and pre-cool at 4℃ for the last 20min centrifuge).

...

Embodiment 3

[0086] Example 3 Using centrifugal collection method to extract sandy soil metagenome

[0087] (1) Weigh 15g of sandy soil sample into a grinding bowl pre-cooled by liquid nitrogen.

[0088] (2) Add liquid nitrogen to grind, continue to add liquid nitrogen to grind before the sample is dissolved, repeat adding three times and grind three times

[0089] (3) Transfer all samples to 50-ml Falcon tubes and add 16.5ml DNA extraction solution. Mix well (measure the pH of the supernatant if it is not 8, adjust to 8.

[0090] (4) Add 40 μl proteinase K (20mg / ml), mix gently by inversion, 37°C, 30min.

[0091] (5) 65°C, +1.83ml 20% SDS, mix well, and incubate for 1-2 hours. Mix every 20 minutes.

[0092] (6) 7000rpm, 15min, collect the supernatant into a new Fecon tube, and store it at 4°C.

[0093] (7) Add 6ml of extract solution, add 0.67ml of 20% SDS, mix well, 65°C, 15min (extract the remaining precipitated sample again).

[0094] (8) 7000rpm for 15min, and mix the supernatan...

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Abstract

The invention discloses a method for extracting soil microbial metagenomic DNA and a corresponding kit. The method for extracting soil microbial metagenomic DNA of the present invention is to pre-treat the soil sample to be tested, effectively avoid the influence of heavy metals, high salinity and low pH on lysozyme, and then lyse the cells twice by adding lysozyme and combining CTAB and SDS , further freeze-thaw treatment breaks up more microbial cell walls, and the DNA molecules in it are dissolved to the maximum extent, ensuring the abundance, fragment integrity and purity of DNA. The extraction method of the invention is simple in operation and strong in practicability, and the extracted DNA can be directly applied to analyze the structure and function of the soil microbial community. The extraction method of the present invention is particularly suitable for extracting soil microbial metagenomic DNA in extreme environments and with extremely low biomass, and a corresponding DNA extraction kit can be prepared according to the extraction method of the present invention, which has good application prospects.

Description

technical field [0001] The invention belongs to the technical field of extracting biological genome DNA, and more specifically relates to a method for extracting soil microbial metagenomic DNA and a corresponding kit. Background technique [0002] An extreme environment refers to an environment characterized by extreme high temperature, low temperature, high pressure, high oxygen, high salinity, high radiation, drought, extreme acidity or alkalinity, and high heavy metal content. Typical extreme environments include volcanoes, deserts, hot springs, salt lakes, submarine hydrothermal vents, polar regions, etc. There are various types of microorganisms in extreme environments, and they are widely distributed on the earth. The study of their life activities has important theoretical research and practical application value. At present, there are two main methods for studying microorganisms, namely, the traditional laboratory pure culture method and the culture-free method. Th...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 艾红霞束文圣叶脉徐卓菲张传伦李猛
Owner 广东美格基因科技有限公司
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