CN102286460B

CN102286460B - Extraction of DNA in Pleurotus eryngii by improved CTAB (cetyltrimethylammonium bromide) method

Info

Publication number: CN102286460B
Application number: CN 201110166772
Authority: CN
Inventors: 阳国秀; 易恢满; 夏志兰; 姬建军
Original assignee: HUNAN YUXIU BIOLOGICAL TECHNICAL Co Ltd
Current assignee: Hunan Guoxiu Food Co., Ltd.
Priority date: 2011-06-21
Filing date: 2011-06-21
Publication date: 2012-12-05
Anticipated expiration: 2031-06-21
Also published as: CN102286460A

Abstract

The invention relates to extraction of DNA in Pleurotus eryngii by an improved CTAB (cetyltrimethylammonium bromide) method. 25-35% anhydrous alcohol is added while adding a mixed solution of Tris-phenol, chloroform and isoamyl alcohol, and the OD260/OD280 values of the extracted DNA are respectively 1.9-2.0, which indicates that the addition of the alcohol is beneficial to removing proteins. Since the alcohol is dissolvable in water and has great affinity for water, the hydration shells of the protein granules can be destroyed, so the proteins precipitate at the isoelectric point; and therefore, the protein precipitate can be removed in the extraction process. When the DNA is subjected to ultraviolet detection by five different methods, the result indicates that only the DNA tape extracted by the invention is the brightest, basically has no degradation trailing phenomenon, and only has few substances detained near the sample charging hole, which indicates that the low-concentration alcohol precipitation method can well remove polysaccharides in the DNA.

Description

A kind of CTAB method of improving is extracted the DNA in the Pleurotus eryngii

Technical field

The present invention relates to the process for extracting of a kind of Pleurotus eryngii DNA.Especially a kind of CTAB method is extracted the DNA of Pleurotus eryngii.

Background technology

In the research of edible mushrooms genetically engineered, the extraction of DNA, separation and purifying are one of important techniques at present.But often be rich in polysaccharide in the edible and medicinal fungi, and many physico-chemical properties of polysaccharide are very similar with nucleic acid, thus in the process for extracting of edible mushrooms DNA its polysaccharose substance how to remove be a stubborn problem; Because polysaccharide can suppress the activity of multiple molecular biology enzymes such as DNA restriction enzyme, T4 dna ligase, TaqDNA polysaccharase, the DNA sample that has therefore polluted polysaccharide can't be used for further molecular biology research.Existing CTAB method is extracted the DNA in the plant gene tissue, all is in the CTAB damping fluid, to add PVP to remove a little polysaccharide; At high density Na ⁺Or K ⁺Under the existence condition, remove polysaccharide through Tris-phenol, chloroform extracting.At present normally through calculating OD ₂₆₀/ OD ₂₈₀Ratio is estimated the purity of DNA, if ratio is lower than in 1.8 explanation DNA samples protein contamination is arranged; The OD of existing CTAB ordinary method ₂₆₀/ OD ₂₈₀Ratio all is lower than 1.8; Only explain through PVP and add and use Tris-phenol to remove a little polysaccharide: chloroform: primary isoamyl alcohol comes the extracting protein remains to have considerable polysaccharide to be mingled in polysaccharide pollution problems when also not having more efficient methods to solve the DNA separation and purification at present with DNA.

Summary of the invention

The object of the present invention is to provide a kind of improved CTAB method to extract the DNA of Pleurotus eryngii.Polysaccharide pollution problems when realizing effectively solving the DNA separation and purification.

The present invention includes following steps:

(1) gets the centrifugal 10min of Pleurotus eryngii mycelium 0.3-0.7g, get deposition, put into mortar; The liquid nitrogen grinding that adds mortar volume about 1/2 becomes fine powder; Place the 10mL centrifuge tube, the CTAB that adds 55-70 ℃ of preheating extracts damping fluid 4mL, the vibration mixing; Be put in water bath heat preservation 0.5-1h in the water-bath of 65 ℃ of constant temperature, once at a distance from 10～20min vibration mixing; Take out centrifugal 10min; Get supernatant, transfer in the corresponding test tube;

(2) absolute ethyl alcohol of adding CTAB volume 25-35% in extracting solution; The mixing solutions that adds 4mLTris-phenol, chloroform, primary isoamyl alcohol again, the Tris-phenol in the said mixing solutions: chloroform: primary isoamyl alcohol=(22～28): (21～26): (1～3); Spinning 10min; Abandon deposition and get supernatant;

(3) in supernatant, add the mixing solutions extracting again of chloroform, primary isoamyl alcohol again, chloroform in the said mixing solutions: primary isoamyl alcohol=(24～28): (1～5);

(4) in supernatant, add the sodium acetate of 0.3～0.5ml 3mol/L, pH is 5.2; Add the Virahol of 3～5ml through-20 ℃ of precoolings again, fully mixing spends the night-20 ℃ of placements; Spinning 10min; Abandon supernatant; Get deposition;

(5) with 70% washing with alcohol deposition 2 times, the ethanol that volatilizees under the room temperature does not have the alcohol flavor until deposition but still keeps moistening; Add 100 μ l TE solution dissolving DNAs; Add 1 μ l50mg/mlRNAse again; At 37 ℃ of water-bath digestion 1h.

Said CTAB extracts damping fluid and comprises 100 mmol/LTris-HCl (pH 8.0), 20 mmol/LEDTA (pH 8.0), 1.4 mol/LNaCl, 3%CTAB, 2%PVP, 2.67% beta-mercaptoethanol.

Advantage of the present invention:

The present invention adds the absolute ethyl alcohol of 25-35%, its OD of the DNA that is proposed in the mixing solutions that adds Tris-phenol, chloroform, primary isoamyl alcohol ₂₆₀/ OD ₂₈₀Value all is between the 1.9-2.0.Explain that the alcoholic acid adding helps proteinic removal.This is because ethanol and water dissolve each other, and is very big to the avidity of water, can destroy the hydration shell of protein particulate, makes protein under isoelectric precipitation, so just can well protein precipitation be removed during extracting.Five kinds of methods with different are carried out the ultraviolet detection result respectively, have only DNA band of the present invention the brightest, totally do not have the degraded conditions of streaking basically, and near the material that well, is detained also seldom; Explain and adopt the low-concentration ethanol precipitator method can remove the polysaccharide among the DNA well.

Embodiment

Below in conjunction with embodiment the present invention is described further.

Embodiment 1:

(1) gets Pleurotus eryngii mycelium pellet 0.5g, add liquid nitrogen (be equivalent to mortar volume 1/2) and grind to form fine powder, place the 10ml centrifuge tube; The 3%CTAB that adds 65 ℃ of preheatings extracts damping fluid 4ml (100 mmol/LTris-HCl (pH8.0), 20mmol/LEDTA (pH8.0), 1.4mol/LNaCl; 3%CTAB, 2%PVP, 2.67% beta-mercaptoethanol); The vibration mixing is put in water bath heat preservation 1h in the water-bath of 65 ℃ of constant temperature, vibrates mixing once at a distance from 10～15min; Take out centrifugal 10min; Get supernatant after centrifugal;

(2) adding is equivalent to add for the first time the absolute ethyl alcohol that CTAB measures 30% volume in supernatant; The mixing solutions that adds 4mLTris-phenol, chloroform, primary isoamyl alcohol again, the Tris-phenol in the mixing solutions: chloroform: primary isoamyl alcohol=25:24:1, centrifugal 10min; Abandon deposition and get supernatant;

(3) in supernatant, add the mixing solutions extracting again of 4mL chloroform, primary isoamyl alcohol again, chloroform in the said mixing solutions: primary isoamyl alcohol=24:1; Centrifugal 10min abandons deposition and gets supernatant;

(4) 3mol/L of adding supernatant 1/10 volume, the Virahol of-20 ℃ of precoolings of the sodium acetate of pH 5.2 and equal-volume, fully mixing spends the night-20 ℃ of placements; Spinning 10min; Abandon supernatant; Get deposition,

(5) with 70% washing with alcohol deposition 2 times, vapor away ethanol under the room temperature, do not have the alcohol flavor until deposition but still keep moistening; Add 100 μ l TE solution dissolving DNAs; Add 0.8-1.5 μ l 50mg/mlRNAse again; In 37 ℃ of water-baths, digest 0.5-1h.

The DNA that extracts is carried out the ultraviolet detection of seven parallel appearance; The result is as shown in table 1

Figure 2011101667729100002DEST_PATH_IMAGE001

It is very complete that 0.8% agarose gel electrophoresis detects genomic dna.

Embodiment 2:

Get Pleurotus eryngii mycelium pellet 0.5g, add liquid nitrogen (be equivalent to mortar volume 1/2) and grind to form fine powder, place the 10ml centrifuge tube; The 3%CTAB that adds 65 ℃ of preheatings extracts damping fluid 4ml (100 mmol/LTris-HCl (pH8.0), 20mmol/LEDTA (pH8.0), 1.4mol/LNaCl; 3%CTAB, 2%PVP, 2.67% beta-mercaptoethanol); The vibration mixing is put in water bath heat preservation 1h in the water-bath of 65 ℃ of constant temperature, vibrates mixing once at a distance from 10～15min; Take out centrifugal 10min; Get supernatant after centrifugal;

In supernatant, add and be equivalent to add for the first time the absolute ethyl alcohol that CTAB measures 25% volume; The mixing solutions that adds 4mLTris-phenol, chloroform, primary isoamyl alcohol again, the Tris-phenol in the mixing solutions: chloroform: primary isoamyl alcohol=26:23:2, centrifugal 10min; Abandon deposition and get supernatant;

The mixing solutions extracting again that in supernatant, adds 4mL chloroform, primary isoamyl alcohol again, chloroform in the said mixing solutions: primary isoamyl alcohol=28:3; Centrifugal 10min abandons deposition and gets supernatant;

The 3mol/L that adds supernatant 1/10 volume, the Virahol of-20 ℃ of precoolings of the sodium acetate of pH 5.2 and equal-volume, fully mixing spends the night-20 ℃ of placements; Spinning 10min; Abandon supernatant; Get deposition,

With 70% washing with alcohol deposition 2 times, vapor away ethanol under the room temperature, do not have the alcohol flavor until deposition but still keep moistening; Add 100 μ l TE solution dissolving DNAs; Add 0.8-1.5 μ l 50mg/mlRNAse again; In 37 ℃ of water-baths, digest 0.5-1h.

Claims

1. a DNA method of improving in the CTAB method extraction Pleurotus eryngii is characterized in that, may further comprise the steps:

Said CTAB extracts the 100mmol/L Tris-HCl that damping fluid comprises pH8.0, the 20mmol/L EDTA of pH8.0,1.4 mol/L NaCl, 3% CTAB, 2% PVP, 2.67% beta-mercaptoethanol;

(2) absolute ethyl alcohol of adding CTAB volume 25-35% in extracting solution; The mixing solutions that adds 4mL Tris-phenol, chloroform, primary isoamyl alcohol again, the Tris-phenol in the said mixing solutions: chloroform: primary isoamyl alcohol=(22～28): (21～26): (1～3); Spinning 10min; Abandon deposition and get supernatant;

(5) with 70% washing with alcohol deposition 2 times, the ethanol that volatilizees under the room temperature does not have the alcohol flavor until deposition but still keeps moistening; Add 100 μ l TE solution dissolving DNAs; Add 1 μ l 50mg/ml RNase again; At 37 ℃ of water-bath digestion 1h.