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Method for rapidly extracting long-fragment DNA from fresh fungus mycelia

A technology of mycelium and long fragments, applied in the field of molecular biology, to achieve the effects of saving experimental time, high extraction rate, and easy annotation

Inactive Publication Date: 2018-12-07
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In view of this, the present invention provides a method for rapidly extracting long fragment DNA from fresh fungal mycelium, which solves the problems in the prior art

Method used

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  • Method for rapidly extracting long-fragment DNA from fresh fungus mycelia
  • Method for rapidly extracting long-fragment DNA from fresh fungus mycelia
  • Method for rapidly extracting long-fragment DNA from fresh fungus mycelia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment one: a kind of method that extracts long fragment DNA rapidly from fresh Beauveria bassiana mycelia, comprises the following steps:

[0029] S101 thalline preparation: get on the 7.5cm plate, cultivate the Beauveria bassiana mycelium of 4d (such as figure 1 ), put it into a centrifuge tube and centrifuge at 12000rpm for 2min to remove 75% of the water, then weigh 0.25g of mycelium, and grind it into powder rapidly in liquid nitrogen (such as figure 2 );

[0030] Lysis of S102 cells: Add 1 mL of 65°C preheated DNA extraction buffer (DNA extraction buffer: 0.1mol / L Tris-HCl (pH8.0), 0.5mol / L NaCl, 0.05mol / L EDTA, the mass concentration is 3% SDS), add 60μL proteinase K (20mg / mL), (such as image 3 ) Shake and mix quickly, place in a water bath at 65°C for 30 minutes, and mix 3 times during this period;

[0031] S103DNA extraction: add 0.33mL 5mol / L KAc, ice bath for 20min, then add 1.5 times the volume of phenol (0.1M Tris saturated phenol, pH>7.5):chlorofo...

Embodiment 2

[0033] Embodiment two: a kind of method for rapidly extracting long fragment DNA from fresh culture Cordyceps militaris mycelium, comprises the following steps:

[0034]S101 cell preparation: Take the Cordyceps militaris mycelia cultured on a liquid shaker for 4 days, put them into a centrifuge tube and centrifuge at 12000rpm for 2 minutes to remove 70% of the water, then weigh 0.25g of the mycelium, and quickly grind it into powder in liquid nitrogen ;

[0035] Lysis of S102 cells: Add 1 mL of 65°C preheated DNA extraction buffer (DNA extraction buffer: 0.1mol / L Tris-HCl (pH8.0), 0.5mol / L NaCl, 0.05mol / L EDTA, the mass concentration is 3% SDS), add 60 μL of proteinase K (20mg / mL), shake quickly and mix well, place in a water bath at 65°C for 30 minutes, and mix 3 times during this period;

[0036] S103DNA extraction: add 0.33mL KAc with a concentration of 5mol / L, and ice-bath for 20min; add 1.5 times the volume of phenol (0.1mol / L Tris saturated phenol, pH>7.5):chloroform:is...

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Abstract

The invention discloses a method for rapidly extracting long-fragment DNA from fresh fungus mycelia. The method comprises the following steps: thallus preparation: extracting fresh cultured fungus mycelia, carrying out centrifugation to remove moisture, weighing the mycelia, and rapidly grinding the mycelia into powder in liquid nitrogen; cell lysis: adding a DNA extracting buffer solution, adding60 mu L of 20mg / mL proteinase K, uniformly mixing, and carrying out water bath at 65 DEG for 30 minutes; DNA extraction: adding 0.33mL of KAc, carrying out water bath for 20 minutes, carrying out extraction by virtue of a mixture of phenol, chloroform and isoamyl alcohol, carrying out centrifugation, extracting supernate, adding absolute ethyl alcohol for precipitation, standing at a low temperature for 30 minutes, carrying out centrifugation, and collecting precipitates; purification: adding RNaseA, processing at 37 DEG C for 30 minutes, respectively extracting by virtue of a mixture of phenol, chloroform and isoamylol in a ratio of 25 to 24 to 1 and a mixture of chloroform and isoamylol in a ratio of 24 to 1, and carrying out low-temperature centrifugation for 5 minutes; and extractingsupernate, adding absolute ethyl alcohol for precipitation, selecting precipitates, rinsing, carrying out air drying, dissolving the precipitates into a TE buffer solution, and carrying out preservation for later use. The DNA amount of filamentous fungi extracted by virtue of the method is increased twice, the concentration of the filamentous fungi reaches 277ng / mu L, the lengths of fragments aremore than or equal to 20000bps, and the filamentous fungi are not degraded and are high in purity.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for rapidly extracting long-segment DNA from fresh fungal mycelium. Background technique [0002] Fungi are lower eukaryotes with a large and diverse species. In recent years, with the improvement of high-throughput sequencing technology, more fungi have completed genome sequencing. The in-depth analysis of genomics has promoted the research of biological evolution, phylogenetics, drug target genes, discovery and function of new genes and new editing methods. The length, concentration, and purity of fungal genomic DNA are directly related to the sequencing, assembly, and annotation of fungal genomes. In the process of library construction and high-throughput sequencing, high-quality and long-fragment fungal genomic DNA can reduce splicing errors, better reflect the original gene regulatory sequence and gene structure, and facilitate the annotation of gene function and e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1017C12Q2521/537
Inventor 耿涛王树昌武华周郭锡杰
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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