DNA extracting method for petroleum microorganism in raw petroleum environmental sample

A technology for environmental samples and extraction methods, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of low DNA yield, difficult extraction, and inability to fully and truly reflect the type and quantity of microorganisms, and achieve high extraction efficiency, consistent with clear effect

Inactive Publication Date: 2014-03-26
KARAMAY JINSHAN PETROCHEMICAL LTD CO
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0003] The present invention provides a DNA extraction method for petroleum microorganisms in crude oil environmental samples, which overcomes the above-mentioned deficiencies in the prior art, and can effectively solve the difficulty of extracting microbial DNA in petroleum Large size, low DNA yield, and can not fully and truly reflect the types and quantities of microorganisms in environmental samples

Method used

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  • DNA extracting method for petroleum microorganism in raw petroleum environmental sample

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Comparison scheme
Effect test

Embodiment 1

[0012] Embodiment 1, the DNA extraction method of petroleum microbes in this crude oil environment sample is carried out as follows: the first step, in the centrifuge tube, add 10 microliters to 50 microliters of crude oil, then add 100 microliters of crude oil in the centrifuge tube filled with crude oil Mix microliters of washing buffer evenly to obtain a mixed solution; in the second step, place the centrifuge tube containing the mixed solution in a cell disruptor for 40 to 60 seconds, and then in a cell with a rotation speed of 12,000 to 16,000 rpm Centrifuge in a centrifuge for 10 minutes to 15 minutes, remove the supernatant to obtain a solid phase; the third step, add 200 μl to 300 μl of extraction cleaning solution to the solid phase, and then add 30 μl to 50 μl of lysozyme and mix evenly, and bathe in water for 5 hours to 10 hours at a temperature of 37° C.; the fourth step, extract the solution after the water bath with a bacterial DNA extraction kit to obtain the D...

Embodiment 2

[0013] Embodiment 2, the DNA extraction method of petroleum microbes in this crude oil environment sample is carried out as follows: the first step, add 10 microliters or 50 microliters of crude oil in the centrifuge tube, then add 100 microliters of crude oil in the centrifuge tube filled with crude oil Mix microliters of washing buffer evenly to obtain a mixed solution; in the second step, place the centrifuge tube containing the mixed solution in a cell disruptor for 40 seconds or 60 seconds, and then in a cell with a rotation speed of 12000 rpm or 16000 rpm Centrifuge in a centrifuge for 10 minutes or 15 minutes, remove the supernatant to obtain a solid phase; the third step, add 200 microliters or 300 microliters of extraction cleaning solution to the solid phase, and then add 30 microliters or 50 microliters of lysozyme and mix evenly, and bathe in water for 5 hours or 10 hours at a temperature of 37° C.; the fourth step, extract the solution after the water bath with a...

Embodiment 3

[0014] Embodiment 3, as the optimization of above-mentioned embodiment, washing buffer solution obtains as follows in embodiment 3, by 10g Na 2 HPO 4 With 1g KH 2 HPO 4 , 50g NaCl and 1.5g KCl, in Na 2 HPO 4 KH 2 HPO 4 . Uniformly, to obtain the washing buffer.

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Abstract

The invention relates to the technical field of extraction of DNA in raw petroleum and particularly relates to a DNA extracting method for a petroleum microorganism in a raw petroleum environmental sample. The method comprises the following steps: firstly, adding 10-50mul of raw petroleum into a centrifugal tube, then, adding 100mul of cleaning buffer solution into the centrifugal tube with the raw petroleum, and uniformly mixing to obtain a mixed solution. The concentration of DNA extracted by using the method is increased by 50% as comparison with the concentration of DNA extracted by using a bacterial DNA extraction kit, the DNA extracted by the invention is remarkably brighter than the DNA extracted by using the bacterial DNA extraction kit, and the obtained DNA is clear in strip and free of trailing so as to prove that the DNA extracting method is used for easily extracting the DNA from the raw petroleum, is higher in extraction efficiency and more capable of accurately, comprehensively and truly showing the kinds and amounts of microorganisms in the raw petroleum as comparison with the bacterial DNA extraction kit.

Description

technical field [0001] The invention relates to the technical field of extracting DNA from crude oil, and relates to a DNA extraction method for petroleum microorganisms in crude oil environmental samples. Background technique [0002] In recent years, with people's emphasis on petroleum microorganisms, especially the in-depth study of microbial oil recovery and microbial gasification, and the improvement of understanding of biological treatment of petroleum wastewater, people began to study the composition, distribution and regularity of microorganisms, but the study of microbial composition and An important prerequisite for its distribution law is the extraction of microbial samples. However, due to the low efficiency of extraction of microorganisms in pure crude oil and microorganisms in residual oil, and the extraction effect is not good, the original composition and function of microorganisms in oil cannot be fully displayed, which makes researchers engaged in this envi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 聂春梅魏利陈爱华方新湘赵燕帕提古丽
Owner KARAMAY JINSHAN PETROCHEMICAL LTD CO
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