Extraction method of soil microbe genome DNA and total RNA
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A technology of soil microorganisms and extraction methods, used in recombinant DNA technology, DNA preparation, etc.
Inactive Publication Date: 2011-02-16
FUJIAN AGRI & FORESTRY UNIV
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However, in the process of extracting soil RNA, in addition to the same difficulties as extracting soil DNA, the ubiquitous RNase poses a great threat to the stability of RNA, which is why there are not many soil RNA extraction products in the international and domestic markets. s reason
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Embodiment 1
[0030] The extraction method of soil microbial genome DNA and total RNA of the present invention, concrete steps are as follows:
[0031] (1) Raw material: fresh sugarcane rhizosphere soil from Fuzhou, Fujian (teaching farm of Fujian Agriculture and Forestry University);
[0032] (2) Removal of impurities in soil samples: Weigh 0.5g of fresh sugarcane rhizosphere soil samples into a 2ml sterile centrifuge tube, add 100μl 1M Tris-HCl (pH=5.5), V 1 μl sterile double distilled water (V 1 =900μl-V 2 ) and V 2 μl 0.2M Al 2 (SO 4 ) 3 Aqueous solution (V 2 The gradient is 60, 90, 120, 150, 180, 210, 240, 270, 300μl, and the test soil is extracted separately), vortexed for 30s; then add 1 / 3 V 2 volume of 4M NaOH solution, and V 3 Volume 0.1M Tris-HCl (pH=8) (V 3 =1300- 4 / 3 V 2 -V 1 ), vortex shaking for 30s, adjust the pH value of the soil solution=8 with 4M NaOH solution, centrifuge at 3500rpm for 2min, remove the supernatant with a pipette gun and calculate the supernat...
Embodiment 2
[0037] (1) Removal of impurities in soil samples:
[0038] Weigh 0.5g rhizosphere soil samples of fresh sugarcane (Fuzhou, Fujian), Rehmannia glutinosa (Jiaozuo, Henan), Radix Pseudostellariae (Zherong, Fujian), tobacco (Kunming, Yunnan) and rice (Fuzhou, Fujian) respectively, and place them in 2ml sterile In the centrifuge tube, add 100μl 1M Tris-HCl (pH=5.5), add V 1 μl sterile double distilled water (V 1 =900μl-V 2 ), join V 2 μl 0.2M Al 2 (SO 4 ) 3 (V 2 =60~300, because the contents of impurities such as humus in rhizosphere soil in different regions or different planting materials are different, according to the steps described in Example 1, the most suitable Al for each test soil is verified. 2 (SO 4 ) 3 volume, so the Al added to the sugarcane soil in this example 2 (SO 4 ) 3 The volume is 150μl, add 75μl to rehmannia soil, add 120ul to heterophylla heterophylla soil, add 120ul to tobacco soil, add 150ul to rice soil), vortex for 30s; add 1 / 3 V 2 Volume 4...
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Abstract
The invention provides a method for effectively extracting crop rhizosphere soil microbe genome DNA and total RNA, which comprises the following steps: firstly, removing humus and other impurities which severely disturb nucleic acid extraction in soil samples by aluminum sulfate; further crushing soil microbe cells by glass beads; adding sodium dodecyl sulfonate (SDS) and LiCl solutions for cracking cells and dissociating nucleic acids; adding the mixture of phenol, chloroform and isoamylol for extracting; and then, depositing the extraction solution by isopropanol and sodium acetate to finally acquire the soil microbe metagenome DNA and total RNA. The method is suitable for extraction of the DNA and total RNA of soil in different regions. The invention establishes an extraction technology which has the advantages of simple and easy operation processes and higher purity of the prepared sample, and can provide an important basis for the research of soil metagenomics.
Description
technical field [0001] The invention relates to a method for extracting soil microbial genome DNA and total RNA, belonging to the field of biotechnology. technical background [0002] Due to the limitations of traditional microbiological research methods and other factors, the development and utilization of microbial resources as the source of life science research and the basis of biotechnology innovation once fell into a stalemate of high-frequency repeated screening, and the continuous development of modern biotechnology has gradually overcome these difficulties. . In the study of modern soil microecology, the genomic DNA of microorganisms in the soil is directly extracted, and on this basis, current advanced molecular biology techniques are used, such as amplified fragment length polymorphism (AFLP) technology, terminal restriction fragment length polymorphism T-RFLP technology, single-strand configuration polymorphism (SSCP), denaturation / temperature gradient gel elect...
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