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A Soil DNA Extraction Method Directly Used to Analyze Microbial Community Structure

A technology of microbial community and extraction method, applied in the field of soil DNA extraction for analyzing microbial community structure, to achieve the effect of simple operation and strong applicability

Inactive Publication Date: 2011-12-07
BEIJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Therefore, it is necessary to develop a fast, practical and effective method suitable for soil microbial DNA extraction, so as to solve the problem that there are many impurities and cell lysis in DNA samples obtained by conventional extraction methods. Problems such as low level and humus pollution

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Weigh 2.0 g of soil sample into a 10 ml centrifuge tube, add 5 ml of 1 M phosphate buffer solution with a pH value of 9.5, vortex for 5 min, then centrifuge at 11000 r / min for 10 min, and discard the supernatant. Repeat washing 2 times, add 5m1 lysate 1 and mix well. Then add 1.5mg of proteinase K and 4g of small glass beads, shake at 50°C for 30min, add 1ml of sodium dodecyl sulfate solution with a concentration of 100g / L, vortex for 5min, shake in a water bath at 65°C for 1h, and Centrifuge at 11000r / min for 10min, and collect the supernatant. Then, 1ml of 10M KSCN solution was added to the collected supernatant, shaken in a water bath at 35°C for 15min, centrifuged at 11000r / min for 10min, and the supernatant was collected. Add the collected supernatant to the centrifugal purification column, then centrifuge at 11000r / min for 10min, and pour off the liquid in the centrifuge tube. Repeat this process until all the supernatant has been purified through the spin colum...

Embodiment 2

[0035]Weigh 2.0 g of soil sample into a 10 ml centrifuge tube, add 5 ml of 1 M phosphate buffer solution with a pH value of 9.5, vortex for 5 min, then centrifuge at 11000 r / min for 10 min, and discard the supernatant. Repeat washing 2 times, add 5ml Lysis Solution 2 and mix well. Then add 1.5mg of proteinase K and 4g of small glass beads, shake at 50°C for 30min, add 1ml of sodium dodecyl sulfate solution with a concentration of 100g / L, vortex for 5min, shake in a water bath at 65°C for 1h, and Centrifuge at 11000r / min for 10min, and collect the supernatant. Then, 1 ml of 10M KSCN solution was added to the collected supernatant, shaken in a water bath at 35° C. for 15 min, centrifuged at 11000 r / min for 10 min, and the supernatant was collected. Add the collected supernatant to the centrifugal purification column, then centrifuge at 11000r / min for 10min, and pour off the liquid in the centrifuge tube. Repeat this process until all the supernatant has been purified through t...

Embodiment 3

[0038] Weigh 2.0 g of soil sample into a 10 ml centrifuge tube, add 5 ml of 1 M phosphate buffer solution with a pH value of 9.5, vortex for 5 min, then centrifuge at 11000 r / min for 10 min, and discard the supernatant. Repeat the washing twice, add 5ml Lysis Solution 3 and mix well. Then add 1.5mg of proteinase K and 4g of small glass beads, shake at 50°C for 30min, add 1ml of sodium dodecyl sulfate solution with a concentration of 100g / L, vortex for 5min, shake in a water bath at 65°C for 1h, and Centrifuge at 11000r / min for 10min, and collect the supernatant. Then, 1ml of 10M KSCN solution was added to the collected supernatant, shaken in a water bath at 35°C for 15min, centrifuged at 11000r / min for 10min, and the supernatant was collected. Add the collected supernatant to the centrifugal purification column, then centrifuge at 11000r / min for 10min, and pour off the liquid in the centrifuge tube. Repeat this process until all the supernatant has been purified through the ...

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Abstract

The invention discloses a soil DNA extraction method directly used for analyzing the microbial community structure, which belongs to the technical field of microbiology and molecular biology. Weigh a certain amount of soil sample into a centrifuge tube, add phosphate buffer, vortex, and centrifuge to separate the washed soil sample. Add lysate to washed soil samples and mix well. Then add proteinase K and small glass beads, add sodium dodecyl sulfate solution after shaking, vortex for 5 minutes, shake in a water bath for 1 hour, and centrifuge to separate the supernatant. The KSCN solution was added to the collected supernatant, and the supernatant was separated by centrifugation after shaking in a water bath for 15 min. Add the collected supernatant to the spin column, then centrifuge to discard the liquid. Add washing solution to the centrifugal purification column, and centrifuge to discard the liquid. Then add the eluent to the centrifugal purification column, place it in a water bath for 2 minutes, collect the liquid in the centrifuge tube after centrifugation, which is the purified soil microbial DNA solution, and store it at -20°C for later use. The invention has the characteristics of simple operation of the extraction method and strong applicability, and the extracted soil microbial DNA can be directly applied to molecular operations to analyze the structure of the soil microbial community.

Description

technical field [0001] The invention belongs to the technical field of microbiology and molecular biology, and in particular relates to a soil DNA extraction method directly used for analyzing microbial community structure. Background technique [0002] The effectiveness of soil bioremediation system operation is based on the diversity and functionality of microbial populations. Therefore, studying the diversity and dynamic changes of soil microbial populations at the genetic level can provide insights into the microbial community structure in soil bioremediation systems. And function analysis provides strong technical support. This requires the extraction of microbial genomic DNA from soil for further molecular manipulation. However, due to the humic acid substances and organic matter contained in the soil, the DNA yield will decrease, especially if the humic acid substances in the soil cannot be removed during the extraction process, it will directly affect the subsequen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 豆俊峰丁爱中
Owner BEIJING NORMAL UNIVERSITY
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