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Method for preparing DNA Marker in nested PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) detection of AMF (Arbuscular Mycorrhizal Fungi) community diversity

A diversity and community technology, used in DNA preparation, recombinant DNA technology, etc., to shorten the experimental time

Inactive Publication Date: 2011-08-10
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, as of now, there is no research on DNAMarker products detected by Nested PCR-DGGE in AMF communities

Method used

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  • Method for preparing DNA Marker in nested PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) detection of AMF (Arbuscular Mycorrhizal Fungi) community diversity
  • Method for preparing DNA Marker in nested PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) detection of AMF (Arbuscular Mycorrhizal Fungi) community diversity
  • Method for preparing DNA Marker in nested PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) detection of AMF (Arbuscular Mycorrhizal Fungi) community diversity

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Experimental program
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Embodiment Construction

[0026] The present invention will be described in further detail below in conjunction with accompanying drawing:

[0027]a. Extraction of soil DNA

[0028] a.1 Weigh 10.0g soil sample into a 50ml sterile centrifuge tube,

[0029] a.2 Add liquid nitrogen to the centrifuge tube, repeat 3 times, each interval is 3min,

[0030] a.3 Transfer the soil to a glass mortar and grind it thoroughly for 10 minutes,

[0031] a.4 Put it into a sterile centrifuge tube, add DNA extraction solution: 0.1mol / L Tris-Hcl, 0.1mol / L EDTA, 0.1mol / L Na 3 PO 4 , 1.5mol / L NaCl, 1% CTAB, pH8.01 3.5ml, adding concentration is 20mg / ml, 50μl proteinase K, purchased from TaKaLa company,

[0032] a.5 Put it into the shaker of Harbin Donglian Instrument Equipment Co., Ltd., 37°C, 225rpm, vibrate for 90min,

[0033] a.6 Take out and add 1.5ml 20% SDS with a 1ml pipette (Eppendorf), bathe in 65°C water for 150min, take out the centrifuge tube every 15min, turn it upside down 3 times,

[0034] a.7 Under norm...

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Abstract

The invention discloses a method for preparing a DNA (Deoxyribonucleic Acid) Marker in a nested PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) detection of AMF (Arbuscular Mycorrhizal Fungi) community diversity. A technical scheme disclosed by the invention comprises the steps of extracting a soil DNA, carrying out DGGE analysis, cloning and transforming, verifying the position of a strip on a DGGE photographic film, making a nested PCR-DGGE Marker of AMF community diversity, carrying out PCR amplification by using NS31-GC and GLoI (Glyoxalase I) as primers, analyzing a result in combination with sequences and naming for use. The invention has an important application value in a Nested PCR-DGGE study of an AMF community structure, is beneficial to reduction of the experiment time, and is especially suitable for experiment process with large experiment sample quantity. According to the invention, a series of DNA Markers for studying the AMF community can be developed so that the nested PCR-DGGE technology is more suitable, faster and more accurate in the study on an AMF molecular community.

Description

technical field [0001] The invention relates to a method for preparing an AMF-specific DNA Marker in denaturing gradient gel electrophoresis (DGGE) molecular detection of an arbuscular mycorrhizal fungi (Arbuscular mycorrhizal fungi, AMF) community, belonging to the field of microbial molecular ecology. Background technique [0002] In the field of life science research, DNA Marker, as a molecular marker, plays an extremely important role in various electrophoresis processes in molecular biology. It is made of a mixture of DNA fragments with different molecular weights. There are many types of DNA Markers currently used in this field, such as DL2000, DGL-2000, DGL-4000 and so on. The use of Marker in the experiment can detect whether the gel concentration is appropriate, whether the electrophoresis conditions are appropriate, shorten the time for exploring experimental conditions, improve efficiency, reduce reagent waste, and easily find the cause of experimental failure. A...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 唐明张海涵陈辉
Owner NORTHWEST A & F UNIV
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