Kit and method for extracting collybia radicata genome DNA (Deoxyribonucleic Acid)

A genome and kit technology, applied in the field of molecular biology, can solve the problems of short DNA fragments, toxic reagents, long time-consuming, etc., and achieve the effects of good integrity, increased binding rate, and shortened extraction time.

Inactive Publication Date: 2018-12-21
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This solves the shortcomings of the existing extraction technology, such as large amount of samples, time-consuming, toxic reagents, and short DNA fragments extracted, so as to meet the needs of molecular biology experiments on precious materials such as rhizomes

Method used

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  • Kit and method for extracting collybia radicata genome DNA (Deoxyribonucleic Acid)
  • Kit and method for extracting collybia radicata genome DNA (Deoxyribonucleic Acid)
  • Kit and method for extracting collybia radicata genome DNA (Deoxyribonucleic Acid)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, adopt the kit of the present invention to extract the genomic DNA of rhizome rhizome mycelium and fruiting body

[0035] The composition of the kit includes:

[0036] (1) Balance Buffer, which contains the following components: 1% Na 2 SiO 3 9H 2 O, 1% NaOH;

[0037] (2) Lysis Buffer, which contains the following components: 5.0M guanidine hydrochloride, 25% isopropanol and 1% PVP K30;

[0038](3) Wash Buffer, containing the following components: 20 mM NaCl, 20 mM Tris-HCl, 85% ethanol, pH 7.5;

[0039] (4) Elution Buffer, containing the following components: 10 mM Tris-HCl, pH 8.5;

[0040] (5) DNA adsorption column.

[0041] Further, the DNA adsorption column is an aluminum silicate ceramic fiber membrane DNA adsorption column.

[0042] Collect respectively the mycelium and the fruiting body of Rhizoma Rhizome under the same culture condition, and concrete steps are as follows:

[0043] (1) Add 200 μL of Balance Buffer to the aluminum silicate ce...

Embodiment 2

[0050] Example 2 Using the kit and method of the present invention to extract genomic DNA from edible fungi

[0051] Kit composition includes:

[0052] (1) Balance Buffer, which contains the following components: 1% Na 2 SiO 3 9H 2 O, 1% NaOH;

[0053] (2) Lysis Buffer, which contains the following components: 5.0M guanidine hydrochloride, 25% isopropanol and 1% PVP K30;

[0054] (3) Wash Buffer, containing the following components: 20 mM NaCl, 20 mM Tris-HCl, 85% ethanol, pH 7.5;

[0055] (4) Elution Buffer, containing the following components: 10 mM Tris-HCl, pH 8.5;

[0056] (5) DNA adsorption column.

[0057] Further, the DNA adsorption column is an aluminum silicate ceramic fiber membrane DNA adsorption column.

[0058] Collect the fruiting body, mycelium, and Agaricus blazei, Cordyceps militaris, and Grifola frondosa fruiting body samples respectively, and use the kit and method of the present invention to extract the genomic DNA of these samples. The specific ste...

Embodiment 3

[0065] Embodiment 3, long root mushroom ITS sequence PCR amplification

[0066] Using general primers ITS-4 / 5 for the ITS fragment of edible fungus, the genomic DNA of the extracted fruiting bodies, mycelia, and dry samples of Rhizoma lanceolata, as well as the fruiting bodies of Agaricus blazei, Cordyceps militaris, and Grifola frondosa were amplified by PCR.

[0067] The primer sequences are:

[0068] ITS-4: 5’-TCCTCCGCTTATTGATATGC-3’

[0069] ITS-5: 5’- GGAAGTAAAAGTCGTAACAAGG-3’

[0070] The reagents used for PCR amplification were purchased from Beijing Quanshijin Biotechnology Co., Ltd., and the reaction system was:

[0071]

[0072] The conditions of the PCR reaction are: 94°C 7min → [94°C 30s → 56°C 30s → 72°C 30s] × 34cycles → 72°C 10min → 4°C.

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Abstract

The invention discloses a kit and method for extracting collybia radicata genome DNA (Deoxyribonucleic Acid). The kit is prepared from the following components: an equilibrium liquid including 1 percent of Na2SiO3.9H2O and 1 percent of NaOH, lysate including 5.0M of guanidine hydrochloride, 25 percent of isopropanol and 1 percent of PVP K30, a cleaning solution including 20 mM of NaCl, 20 mM of Tris-HCl and 85 percent of ethanol with pH of 7.5, eluent including 10 mM of Tris-HCl with the pH of 8.5 and a DNA adsorption column. The reagent for extracting the collybia radicata genome DNA does notcontain toxic substances such as phenol and chloroform, so that higher safety is realized; the extraction method is simple and efficient and the extraction time is shortened to 15 minutes; an extracted product has high purity and good integrity, and can be directly used for molecular biology and genetic research.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a kit and a method for extracting the genomic DNA of Rhizome lanceolata. Background technique [0002] DNA is the carrier of genetic information in living organisms, including the genetic traits of organisms. High-quality, high-purity total DNA is the primary condition to ensure the development of molecular biology research such as PCR amplification, restriction endonuclease digestion, genetic map analysis, molecular hybridization, genetic diversity analysis, and genomics. Therefore, it is extremely important to efficiently and rapidly prepare total DNA samples with high purity and good integrity. [0003] At present, the traditional methods for preparing fungal total DNA include CTAB method, SDS method, alkali lysis method and conventional kit method, etc. CTAB method and SDS method use ionic surfactants to lyse fungal cells to release the genome, and then...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor 刘朋虎张煜隆黄在兴胡应平罗海凌苏德伟李晶翁伯琦林占熺
Owner FUJIAN AGRI & FORESTRY UNIV
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