Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for efficiently extracting underground water microbial DNA for PCR amplification

A technology for microorganisms and groundwater, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problems of small number of microorganisms in groundwater and high extraction difficulty, and achieve the effect of simple method, low cost and low operation cost

Inactive Publication Date: 2013-10-02
INNER MONGOLIA UNIV OF SCI & TECH
View PDF3 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the number of microorganisms in groundwater is small and the extraction is difficult. It is a huge challenge to extract high-quality microbial genome DNA from groundwater simply and effectively.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for efficiently extracting underground water microbial DNA for PCR amplification
  • Method for efficiently extracting underground water microbial DNA for PCR amplification
  • Method for efficiently extracting underground water microbial DNA for PCR amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The technical solution adopted by the present invention is a method for efficiently extracting groundwater microbial DNA for PCR amplification. The method includes the steps of collecting groundwater microorganisms, cracking groundwater microbial cells, purifying cell lysates, and extracting and purifying microbial genome DNA. The concrete steps of the inventive method are:

[0042] The first step is to collect groundwater microorganisms: take groundwater samples and filter them with sterile microporous membranes to collect microorganisms, and then use a buffer containing cetyltrimethylammonium bromide to elute the microorganisms on the sterile microporous membranes Microbial cell liquid is obtained; the buffer contains cetyltrimethylammonium bromide, which can achieve better elution effect.

[0043] The second step, cracking groundwater microbial cells: add lysozyme to the microbial cell liquid for lysozyme enzymolysis, transfer the lysate after lysozyme enzymolysis in...

Embodiment 2

[0047] On the basis of embodiment 1, the present invention is in order to improve the extraction effect of genomic DNA, preferred embodiment also has, and the operation process of cracking groundwater microbial cell described in the second step is:

[0048] ①Lysozyme enzymatic hydrolysis: add lysozyme solution to the microbial cell liquid until the concentration of lysozyme in the microbial cell liquid is 10mg / mL, put in a water bath at 35°C for 1.5h, and then transfer to a new centrifuge tube;

[0049] ②Protease hydrolysis: add proteinase K to the microbial cell fluid after lysozyme hydrolysis, until the concentration of proteinase K in the microbial cell fluid is 0.2mg / mL, then add 100 microliters of dodecyl sulfate with a mass concentration of 20% Sodium, 40 microliters of 5mol / L NaCl solution and 20 microliters of 2mol / L NaCl 3 PO 4 The solution was placed in a water bath at 53° C. for 2 hours, centrifuged at a speed of 11,000 r / min for 5 minutes, and the supernatant was ...

Embodiment 3

[0052] On the basis of Example 2, the present invention obtains high-purity genomic DNA in order to purify the cell lysate, and a preferred embodiment also has the operation process of purifying the cell lysate described in the third step as follows:

[0053] ① Add solution III which is 0.2 times the volume of the cell lysis supernatant to the cell lysis supernatant and mix evenly. After 12 minutes in ice bath, centrifuge at 12,000 r / min for 13 minutes at 4°C to remove the precipitate and obtain No. 1 supernatant;

[0054] ② Add pre-cooled absolute ethanol twice the volume of the No. 1 supernatant to the No. 1 supernatant, precipitate at -20°C for 60 minutes, then centrifuge at 12,000 r / min at 4°C for 14 minutes, and remove the supernatant After obtaining No. 1 precipitation;

[0055] ③ Add 500 microliters of extraction buffer to the No. 1 precipitate to dissolve the No. 1 precipitate to obtain a crude DNA extraction solution. Using absolute ethanol to precipitate DNA and RNA...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
pore sizeaaaaaaaaaa
Login to View More

Abstract

The invention discloses a method for efficiently extracting underground water microbial DNA for PCR amplification. The method comprises the following steps: 1, collecting underground water microbes, collecting microbes by using an aseptic millipore filter, and eluting with a buffer solution containing cetyltrimethylammonium bromide to prepare a microbial cytochylema; 2, disrupting underground water microbial cells, adding lysozyme and protease K to the microbial cytochylema, carrying out warm water bathing, and centrifuging to obtain a cell lysis supernatant; 3, purifying the obtained lysate, and carrying out centrifuging purification of the obtained cell lysis supernatant a plurality of times to obtain a crude DNA solution; and 4, purifying microbial genome DNA, carrying out centrifuging purification of the crude DNA solution, and extracting with ethanol having a volume concentration of 70% to obtain purified microbial genome DNA. The method has the advantages of efficient extraction of the high-quality underground microbial genome DNA, simple operation and high extraction purity.

Description

technical field [0001] The invention relates to a method for extracting genomic DNA, in particular to a method for efficiently extracting groundwater microbial DNA used for PCR amplification. Background technique [0002] Due to the massive discharge of factory waste water, domestic waste water and domestic garbage, and the extensive use of chemical fertilizers and pesticides, groundwater pollution has become increasingly serious. At present, 90% of the groundwater in the country has been polluted to varying degrees, of which 60% are seriously polluted. Water pollution is on the rise. Threats to the living conditions and even lives of all human beings. It has become an important topic in today's society to timely detect whether groundwater is polluted and the degree of pollution, and how to control water pollution. [0003] Groundwater microorganisms can scientifically evaluate the quality changes of groundwater, but the microorganisms that can be cultivated in the laborato...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 张雪峰郑春丽王建英杨彪姚卫华高晓玲林忠吕保义
Owner INNER MONGOLIA UNIV OF SCI & TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com