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Method and kit for extracting total desoxyribonucleic acid in crude oil

A total deoxyribonucleic acid and kit technology, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of difficulty in efficient DNA extraction, large differences in crude oil density, and few new oil reservoirs, etc., to increase the cracking effect and efficient extraction. Protocols, Effects of Increased Yield and Purity

Active Publication Date: 2017-05-31
CHINA UNIV OF PETROLEUM (BEIJING)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are not many new oil reservoirs in my country at this stage, and most of them are old oil reservoirs in the middle and late stages of development. Different flooding operation methods lead to a large amount of suspended matter in the produced fluid, making it difficult to filter and separate samples
In particular, crude oil is a complex system consisting of a large number of hydrocarbons and non-hydrocarbon compounds (a. Du Weidong, Wan Yunyang, Zhong Ningning, etc. Current status of oil pollution in soil and sediments [J]. Journal of Wuhan University (Natural Science Edition). 2011(04):311-322; b.Du W, Wan Y, Zhong N, et al.Status quo of soil petroleum contamination and evolution of bioremediation[J].Petroleum Science.2011,8(4):502-514. ), cannot fully diffuse in water, resulting in the inability of microorganisms in crude oil to fully mix with water. For a long time, no one has paid attention to it. It is even believed that microorganisms cannot exist in crude oil, and the density of different types of crude oil varies greatly. These characteristics give DNA high efficiency. Extraction poses considerable difficulty
At present, for the extraction of the total DNA of crude oil samples from new and old oil reservoirs, a general extraction and purification method system has not been formed at home and abroad. Therefore, it is urgent to build a general purification system in the field of oil reservoirs.

Method used

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  • Method and kit for extracting total desoxyribonucleic acid in crude oil
  • Method and kit for extracting total desoxyribonucleic acid in crude oil
  • Method and kit for extracting total desoxyribonucleic acid in crude oil

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Take 100mg of L2YHC23 heavy crude oil sample, pretreat with isooctane, lyse live cells and / or dead cells by positive-negative surfactant method, and purify by four methods, namely chloroform-alcohol precipitation method, phenol / Chloroform-alcohol precipitation method, phenol / chloroform-purification column method and chloroform-purification column method.

[0051] The pretreatment method in this example: add an equal volume of isooctane and an appropriate amount of glass beads to the L2YHC23 oil sample, vortex for 20 minutes, and grind thoroughly to form a homogenate. After loading into a centrifuge tube, centrifuge (8000 rpm, 20° C., 20 min), discard the supernatant, and keep the precipitate at the bottom of the centrifuge tube.

[0052] In this embodiment, the positive-negative surfactant method is used to lyse living cells and / or dead cells: take 5 g of the pretreated sample in a centrifuge tube, add 150 μL of lysozyme with a concentration of 20 mg / mL, shake, and add...

Embodiment 2

[0063] Compared with Example 1, in Example 2, the pretreatment method was replaced by TE method, and 10 mg of L2YHC23 crude oil sample was taken and pretreated with TE. The cracking process was completely consistent with Example 1, and the purification method was also divided into (a) chloroform-alcohol Precipitation method, (b) phenol / chloroform-alcohol precipitation method, (c) chloroform-purification column method, (d) phenol / chloroform-purification column method, the purification process is exactly the same as that of Example 1.

[0064] The specific steps of the pretreatment method in this example: add 10mL TE and an appropriate amount of glass beads to the L2YHC23 crude oil sample, vortex for 10-20min, and fully grind to form a homogenate. After loading into a centrifuge tube, centrifuge (8000 rpm, 20° C., 20 min), discard the supernatant, and keep the precipitate at the bottom of the centrifuge tube.

[0065] The results of DNA extraction are shown in Table 2:

[0066]...

Embodiment 3

[0070] Take 100 mg of L2YJC226 medium crude oil sample, pretreat it with isooctane, and extract the total DNA of the crude oil by positive-negative surfactant method + chloroform-alcohol precipitation method. The operation process and method are exactly the same as those in Example 1.

[0071] The results of DNA extraction are shown in Table 3:

[0072] The concentration and purity of table 3 embodiment 3 extraction liquid DNA

[0073]

[0074] The detection results are shown in Table 3. The total DNA of crude oil can be effectively extracted by using the positive-negative surfactant method+chloroform-alcohol precipitation method of the present invention.

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Abstract

The invention relates to a method and a kit for extracting total desoxyribonucleic acid in crude oil. The method comprises the following steps of carrying out pretreatment on the crude oil; cracking a viable cell and / or a dead cell by a positive-negative surfactant method to obtain a supernatant fluid containing the total desoxyribonucleic acid; extracting the supernatant fluid to obtain the total desoxyribonucleic acid by utilizing one of four purification methods of chloroform-alcohol precipitation, phenol / chloroform-alcohol precipitation, a chloroform-purification column and a phenol / chloroform-purification column. According to the method and the kit, aiming at the self property of the crude oil and the characteristic that components are complicated, four methods and kits for extracting the total desoxyribonucleic acid in the crude oil, which are wide in scope of application, are developed; the total desoxyribonucleic acid in the crude oil can be effectively extracted.

Description

technical field [0001] The invention relates to a kit and a method for extracting total deoxyribonucleic acid (DNA) from crude oil, and belongs to the technical field of microbial geology. Background technique [0002] Efficient extraction of total DNA from the reservoir environment is the premise and basis for the application of molecular biology methods to study the microbial diversity of reservoirs, and the quality of sample DNA is the key factor for the success of subsequent molecular biology experiments. Whether it is a newly developed or developing reservoir (respectively referred to as new reservoir and old reservoir), the produced fluid samples include crude oil, oil-water mixture, formation water and solid matter, mostly oil-water mixture. There are not many new oil reservoirs in my country at this stage, and most of them are old oil reservoirs in the middle and late stages of development. Different flooding operation methods lead to a large amount of suspended matt...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 万云洋穆红梅洪宁顾雪莹田燕
Owner CHINA UNIV OF PETROLEUM (BEIJING)
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