PCR-DGGE (Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis) analysis method of microbial diversity in bioremediation process of soil polluted by petroleum

A technique of PCR-DGGE and soil biology, applied in the field of soil microbial community diversity analysis, to achieve high purity and sufficient yield

Inactive Publication Date: 2011-09-07
BEIHANG UNIV
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Problems solved by technology

[0004] Although ideal experimental results can be obtained by strictly referring to the operating instructions and related references, due to the wide variety of samples available for research, and PCR-DGGE technology for different samples, it is necessary to determine the relevant important reagent dosage and Grasp some experimental details, and most literatures do not specify the dosage of key reagents, so it is necessary to give the best conditions that have been explored for typical samples, in order to provide technical guidance for follow-up research, and avoid cumbersome trials of a large number of experimental conditions

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  • PCR-DGGE (Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis) analysis method of microbial diversity in bioremediation process of soil polluted by petroleum
  • PCR-DGGE (Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis) analysis method of microbial diversity in bioremediation process of soil polluted by petroleum
  • PCR-DGGE (Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis) analysis method of microbial diversity in bioremediation process of soil polluted by petroleum

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Embodiment Construction

[0019] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

[0020] 1. Oil-contaminated soil samples and their pretreatment

[0021] PCR-DGGE analysis was performed on the soil in the early stage of remediation (15 days) to investigate the microbial community structure and dynamic changes in the soil, as well as the microbial diversity in the system.

[0022] The soil used for the small test of oil-contaminated soil bioremediation was taken from an oil well in Shengli Oilfield. After removing impurities, air-drying, breaking up, passing through a 200-mesh sieve, and mixing, the soil was taken and divided into 10 small flower pots. , and screen the domesticated strains after mutagenesis by adding heavy ions. The basic physical and chemical properties of the oil-contaminated soil samples are shown in Table 1. The oil content of the soil is as high as 194.5g / (kg dry soil), far exceeding the critical value of 500m...

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Abstract

The invention belongs to the technical field of soil microbial community diversity analysis and discloses optimal conditions and reagent dosage in a PCR-DGGE (Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis) method used for analyzing microbial community diversity of soil polluted by petroleum. The analysis method comprises the following steps of: 1, extracting enough DNA from 0.4g of soil samples; 2, carrying out PCR (Polymerase Chain Reaction) on 1 microliter of purified DNA; 3, performing a DGGE (denaturing gradient gel electrophoresis) experiment on 3-5 microliters of PCR products; 4, adding 9 microliter of TEMED (tetramethylethylenediamine) and 40 microliter of 10% APS (ammonium sulfate) into 18ml of modified acrylamide glue solution for preparing gel; 5, filling the gel, covering the top with a preservative film, standing still overnight to finally obtain excellent modified gel; 6, performing electrophoresis for 5 hours at the voltage of 120V and the electrophoresis temperature of 60 DEG C to obtain a good DGGE map; and 7, performing silver staining, wherein a stop solution is not used necessarily. The method is mainly used in the molecular ecology researches in the bioremediation process of soil polluted by petroleum, and also can provide references for molecular ecology researches of other environmental samples.

Description

technical field [0001] The invention belongs to the technical field of soil microbial community diversity analysis, and is specifically aimed at the PCR-DGGE analysis method of microbial diversity in the process of bioremediation of oil-contaminated soil, that is, the optimal PCR-DGGE experimental conditions are obtained by improving and optimizing the microbial community diversity PCR-DGGE experimental conditions. Optimum reagent dosage and experimental conditions. The invention is mainly applied to molecular ecology research in the bioremediation process of petroleum-contaminated soil, and can also provide references for molecular ecology research on other environmental samples. Background technique [0002] Petroleum, as an important industrial energy source, plays a major decisive role in economic development, but at the same time, in the process of production, transportation, storage and use of petroleum, it has caused serious pollution to the natural environment, espec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N27/447
Inventor 杨玉楠郭若勤
Owner BEIHANG UNIV
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