Method for extracting prawn intestinal microbial DNA

A technology of intestinal microorganisms and extraction methods, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve problems such as difficult to obtain DNA samples, and achieve the effect of strong specificity, good versatility, and large concentration

Inactive Publication Date: 2010-06-23
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult to obtain DNA samples that can be used for amplification according to the conventional method of extracting in

Method used

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  • Method for extracting prawn intestinal microbial DNA
  • Method for extracting prawn intestinal microbial DNA

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Take Penaeus vannamei as an example; put the shrimp intestines into absolute ethanol and freeze at -80°C; take 0.02 g of shrimp intestines, grind them under liquid nitrogen, and add a total of 1 mL of PBS and 20 μL of 20% (volume ratio) PVPP to homogenize slurry, 250 μL of 0.5% (volume ratio) Tween-80, eluted by pipetting for 3 min, the PBS solution, the composition of which is 2 mM KH 2 PO 4 , 10mM Na 2 HPO 4 , 137 mM NaCl, 2.7 mM KCl, pH=7.4. Then centrifuge at 200g for 6min, take the supernatant for later use, add 1 mL of PBS to the precipitation, then take the supernatant after centrifugation at 200g, combine the two supernatants; centrifuge the combined supernatant at 300g for 6min , take the supernatant; the obtained supernatant is then centrifuged at 1200g for 6 min, the supernatant is collected, and washed twice with PBS; 130 μL of CTAB extract and 10 μL of lysozyme are added to the obtained supernatant, and placed in 37 Shake for 30 min at °C; then add 15 μ...

Embodiment 2

[0025] Take Chinese prawn as an example, put the shrimp intestine into absolute ethanol and freeze at -80°C; take 0.05g of shrimp intestine, grind under liquid nitrogen, add a total of 0.8mL PBS and 15μL 20% (volume ratio) PVPP Homogenate, 200 μL of 0.5% (volume ratio) Tween-80, eluted by pipetting for 5 min, the PBS solution, the composition of which is 2 mM KH 2 PO 4 , 10mM Na 2 HPO 4, 137 mM NaCl, 2.7 mM KCl, pH=7.4. Then centrifuge at 200g for 10min, take the supernatant for later use, add 0.8mL PBS to the precipitation, take the supernatant after centrifugation at 200g, combine the two supernatants; centrifuge the combined supernatant at 300g 10min, take the supernatant bacterial liquid; the obtained supernatant bacterial liquid is centrifuged at 1200g for 10 min, collect the supernatant bacterial liquid, and wash with PBS 3 times; add 100 μL CTAB extract and 15 μL lysozyme to the obtained supernatant bacterial liquid, put in Shaker at 37°C for 40min; then add 20μL of...

Embodiment 3

[0027] Taking the Japanese prawn as an example, take the shrimp intestine and put it in absolute ethanol and freeze it at -80°C; take 0.01g of the shrimp intestine, grind it under liquid nitrogen, add a total of 1.2mL PBS and 25μL 20% (volume ratio) PVPP to make it uniform. slurry, 300 μL of 0.5% (volume ratio) Tween-80, eluted by pipetting for 4 min, the PBS solution, the composition of which is 2 mM KH 2 PO 4 , 10mM Na 2 HPO 4 , 137 mM NaCl, 2.7 mM KCl, pH=7.4. Then centrifuge at 200g for 8min, take the supernatant for later use, add 1.2mL PBS to the precipitation, take the supernatant after centrifugation at 200g, combine the two supernatants; centrifuge the combined supernatant at 300g 8 min, take the supernatant liquid; the obtained supernatant liquid is centrifuged at 1200g for 8 min, collect the supernatant liquid, and wash it twice with PBS; add 150 μL CTAB extract and 12 μL lysozyme to the obtained supernatant bacterial liquid, put it in Shake at 37°C for 35min; t...

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Abstract

The invention relates to a method for extracting prawn intestinal microbial DNA, and belongs to the fields of microbiology and molecular ecology. The method comprises the following steps of: grinding by using liquid nitrogen; breaking cell walls by using mechanical force; cracking cells by adopting lysozyme and SDS lysate; obtaining DNA deposit through separation and precipitation; and finally dissolving the DNA deposit with TE to obtain the DNA with good quality. The product DNA fragment obtained by the method is greater than 20Kb; and can be subjected to gene magnification and sequencing. The method has good generality and is used for extracting the intestinal microbial DNA of various crustacean aquatic animals. High-quality template DNA can be obtained through extraction at one time without repeated experiment; and the obtained DNA can be applied to the research of molecular ecology, system evolution and the like.

Description

technical field [0001] The invention belongs to the field of microbiology and molecular ecology, and particularly relates to a method for extracting prawn intestinal microorganism DNA. Background technique [0002] The research on gut microbes has become a hotspot. At present, most researches on the microbial diversity of shrimp gut are culturable bacteria, but more than 99% of the microorganisms in nature are not culturable. An in-depth understanding of the microbial community structure of the shrimp gut requires the establishment of molecular methods to study the microbial composition of the shrimp gut. Gut microbial DNA is a prerequisite for molecular studies. Whether a high DNA extraction rate and high-quality DNA can be obtained, so as to truly reflect the actual situation of the microbial community, is the key to ensuring the reliability of the research results. How to obtain high-quality and relatively complete genomic DNA of intestinal flora is the key to the resea...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 刘淮德王雷刘梅王宝杰蒋克勇
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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