Small quality fast extraction method for soil total DNA

An extraction method and soil technology, applied in the field of microbiology and molecular biology, can solve the problems of difficult separation of cells, uneconomical extraction, general and other problems

Inactive Publication Date: 2007-07-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The indirect method of extracting total DNA from soil is to separate the cells first and then lyse. Because many microorganisms are closely combined with soil particles, the cells are not easy to separate, resulting in low final DNA yield and very time-consuming. This method is generally seldom used; the direct method is direct Cells are lysed from soil, which is time-consuming and ineffective; the kit method is simple and fast, but expensive and uneconomical for the extraction of a large number of samples

Method used

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  • Small quality fast extraction method for soil total DNA
  • Small quality fast extraction method for soil total DNA
  • Small quality fast extraction method for soil total DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Total DNA Extraction from Orthic Anthrosols

[0028] The experimental soil was taken from the surface soil sample of the middle school affiliated to Huazhong Agricultural University in Wuhan City, Hubei Province, China (the soil is classified as dry-cultivated soil, pH=5.87, and named as Orthic Anthrosols). Sieve and store at -20°C until use. The specific operation steps are as follows:

[0029] (1) Making cracking tubes: Weigh 10 grams each of quartz sand and silica powder, wash with dilute nitric acid first, then wash with deionized water and soak overnight, then dry at 80°C, and accurately weigh the dried Each 0.3g of quartz sand and silicon dioxide powder is put into a 2ml centrifuge tube, and sterilized at 121°C for 30 minutes;

[0030](2) Weigh 0.5 grams of soil sample into the lysis tube of step (1), add lysis buffer (preparation of lysis buffer: 0.1M Tris-HCl, 0.1M EDTA, 0.1MH 3 PO 4 Buffer, 1.5M NaCl, pH8.0, sterilized at 121°C for 30 minutes, t...

Embodiment 2

[0042] Embodiment 2: PCR detection is done to the DNA extracted in embodiment 1

[0043] The DNA extracted in Example 1 was used as a template, and the designed primers: 338F 5' CTCCTACGGGAGGCAGCAG3' and 1492R5'GGTTACCTTGTTACGACTT3' were used to amplify soil microbial 16S rDNA (16S ribonucleosome RNA gene). The 16S rDNA of soil microorganisms were amplified by the indirect method, the kit method and the total soil DNA prepared by the method of the present invention without dilution and diluted 10 times as templates for PCR reactions. The PCR reaction system is as follows (50ul): ddH 2 O: 34.5ul; DNA: 2ul; 10×PCR buffer (TaKaRa): 5ul; 10×MgCl 2 (TaKaRa): 5ul; dNTP (Promega, 10mM each): 1ul; 16S rDNA primer 338F (50ng / ul): 1ul; 16S rDNA primer 1492R (50ng / ul): 1ul; Taq Enzyme (TaKaRa, 5U / ul) : 0.5ul. The PCR program is as follows: pre-denaturation: 94°C for 5 minutes; denaturation: 94°C for 45 seconds; annealing: 49°C for 45 seconds; extension: 72°C for 1.5 minutes; final ext...

Embodiment 3

[0045] Example 3: Enzyme digestion detection of DNA extracted using the method of the present invention

[0046] EcoR I (G↓AATTC) and PstI (CTGCA↓G) double enzyme digestion is done to the DNA extracted by the method of the present invention in Example 1 (references: J. Sam Brook, D.W. Russell. Molecular Cloning Experiment Guide . Third Edition. Science Press. 2003). Enzyme digestion system (20ul): ddH 2 O: 11ul; 10×H buffer (TaKaRa): 2ul; EcoR I (TaKaRa, 15U / ul): 1ul; PstI (TaKaRa, 15U / ul): 1ul; DNA: 5ul. Digestion conditions: overnight at 37°C. As can be seen from Fig. 3, the DNA after enzyme digestion presents a continuous band shape, which proves that the DNA is cut into small fragments in a smear shape, indicating that the DNA extracted by the method of the present invention can meet the requirements of enzyme digestion.

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Abstract

The invention discloses a method for fast extracting the total soil DNA. It takes soil as raw material, shaking with quartz sand and silicon dioxide powder, cracking with cetyltrimethylammonium bromide for DNA releasment, the released DNA can be absorbed by diatomite under weak acid or netural pH condition and high concentration thiocyanate ion, and the DNA will be eluted by low salty buffer solution or deionized water under weak alkaline condition. The extracted DNA with enough purity and amount can be used for PCR and endonuclease reaction. The main steps comprise cell cracking, DNA extraction and DNA purification. The invention is characterized by the simple, fast and easy method, and high purity and low cost extracted DNA. The extracted DNA can be used for kit production. The soil total DNA can be extracted in half hour for microbiological and molecular biological research.

Description

technical field [0001] The invention belongs to the technical field of microbiology and molecular biology, and in particular relates to a method for rapidly extracting total DNA from soil in a small amount. Background technique [0002] The method that is usually used for soil total DNA extraction mainly contains indirect method at present (Andrew E.Berry, Claudia Chiocchini, TinaSelby, Margherita Sosio, Elizabeth M.H.Wellington.(2003) Isolation of high molecular weight DNA from soil forcloning into BAC vectors.FEMS Microbiology Letters.223:15-20), direct method (Zhou, J., Mary Ann Bruns, M.A., and Tiedje, M.J. (1996) DNA Recovery from Soils of Diverse Composition.Applied and Environmental Microbiology62(2):316-322) and Kit method (FastDNA SPIN Kit for Soil of Q.BIOgene Company), Chinese Invention Patent Application Publication (Publication No. CN1456684A, Patent Application No.: 03120964.5) discloses "a primer design scheme for PCR-DG...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 王革娇蔡林
Owner HUAZHONG AGRI UNIV
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