CN102433322A - Extraction method for honey gene - Google Patents
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- CN102433322A CN102433322A CN2011104019357A CN201110401935A CN102433322A CN 102433322 A CN102433322 A CN 102433322A CN 2011104019357 A CN2011104019357 A CN 2011104019357A CN 201110401935 A CN201110401935 A CN 201110401935A CN 102433322 A CN102433322 A CN 102433322A
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Abstract
The invention discloses an extraction method for a honey gene and relates to the field of biological chemistry. According to the extraction method, more than 10ml of honey is adopted for extracting; soluble polysaccharides in the honey are removed by adopting a phosphate buffered solution; plant component cells in the honey are cracked by using a cracking buffered solution; meanwhile, mostly glucoprotein in sodium chloride solution with high concentration is removed, so that the interference of the glucoprotein on DNA (Deoxyribonucleic Acid) during purification is reduced; protein is further removed by using a mixed solution of phenol, chloroform and isoamylol; and finally, the DNA is precipitated by using isopropanol under the low-temperature condition so as to obtain purified honey DNA. The extraction method disclosed by the invention has the advantages of stable result, favorable repeatability, simpleness in operation, low expense and no need of special instrument or reagent; the obtained honey DNA has higher quality and can meet the requirement of a polymerase chain reaction; and quick identification of the quality of the honey can be realized. According to the extraction method disclosed by the invention, a honey gene extraction kit can be developed and the obtained honey DNA can be extracted and applied to quick identification of a honey source as a reagent.
Description
Technical field
The present invention relates to biochemical field, be specifically related to basis of honey because of process for extracting.
Background technology
Honey is nectar, secretory product or the honeydew of honeybee herborization, after self secretory product combines, through fully brewageing the crude sweet material that forms.Containing multiple sugar in the honey, mainly is fructose and glucose.In addition, also contain organic acid, enzyme and solid particulate matter such as plant pollen etc.Because the honey market price is higher, the huge market demand has some illegal retailers to mix the spurious with the genuine, adulterate, and has seriously damaged consumer's interests, therefore is badly in need of setting up the Identification Evaluation system of honey product.
Though honey product is difficult to from aspects such as outward appearance, mouthfeels distinguish, honey DNA is relevant with nectariferous plant, and this can't forge, and therefore, the gene test means are seeming particularly important aspect the quality control of honey.Kind component detection method commonly used both at home and abroad at present mainly is to be the polymerase chain-reaction test method on basis with nucleic acid, is called for short the PCR method.The concentration of PCR method general requirement template DNA reaches more than the 20 ng/ μ l, and purity adopts OD
260/ OD
280Ratio is represented, OD
260/ OD
280Ratio needs between 1.7 ~ 2.0.Because the honey ingredient is complicated, DNA is difficult to be extracted, and this has limited carrying out of honey PCR detection method to a certain extent.
DNA extraction method commonly used at present mainly contains high salt low pH method, phynol method and CTAB method.
Wherein high salt low pH method is mainly used in the extraction of plant tissue DNA.Low pH helps preventing the polyphenols oxidation in the plant, and high salt then is to help protein precipitation.This experiment is attempted high salt low pH method is applied in the extraction of honey DNA, through measuring, adopts concentration and the purity of the acacia honey DNA that high salt low pH method extracts lower, and the concentration of DNA is 13ng/ μ l, OD
260/ OD
280Be 1.25, be difficult to satisfy the requirement of PCR reaction.Through analyzing, the major cause that high salt low pH method is not suitable for the honey DNA extraction is to compare with general plant tissue, and the dna content in the honey sample is less, adopts high salt low pH method to be difficult to reach the purpose of enriching and purifying honey DNA.
Phynol method is to adopt lysate that DNA is discharged, and alternately carries out extracting with phenol, these two kinds of different protein denaturants of chloroform then, removes the protein in the solution.The strong and ability of removing polysaccharide of the ability of phenol, chloroform deproteinize a little less than, be applicable to the extraction of the DNA of animal tissues.This experiment is attempted phynol method is applied in the honey DNA extraction, and after the repeatedly extracting of sample through phenol and chloroformic solution, the quality of the DNA that is obtained is lower, and the concentration of DNA is 11ng/ μ l, OD
260/ OD
280Be 1.43, be difficult to satisfy the requirement of PCR reaction.The major cause that phynol method is not suitable for the honey DNA extraction is that the dna content in the honey sample is low; Loss is bigger in the repeatedly extractive reaction of phenol and chloroformic solution; Cause the final dna content that obtains on the low side, the DNA quality does not reach the requirement that next step PCR detects.
The CTAB method is because can remove polysaccharide impurity, so be usually used in the extraction of plant tissue DNA preferably.CTAB, promptly cetyl trimethylammonium bromide is a kind of cationic detergent.CTAB both can the lysing cell film, and nucleic acid is discharged, and can under different salt ionic concentrations, optionally precipitate polysaccharide or nucleic acid again, thereby reach the purpose of purify DNA.Present method attempts conventional CTAB method is directly applied in the extraction of honey DNA, and the concentration of gained honey DNA is 16ng/ μ l, OD
260/ OD
280Be 1.39, can not satisfy the demand that follow-up PCR detects.The major cause that conventional CTAB method is not suitable for the honey DNA extraction is that honey component is complicated, and particularly polysaccharide and protein content are high, use conventional CTAB method and can't effectively remove polysaccharide and albumen impurity, have influenced yield and the purity of honey DNA.
Because honey component is complicated, wherein about 75% composition is a carbohydrate, also contains plurality of enzymes, amino acid, inorganic salt etc. simultaneously, and conventional CTAB method also can't be extracted and obtain high density and highly purified honey DNA.So must improve, searching can be extracted the method for high density and high purity honey DNA, to satisfy the requirement that PCR detects.
Summary of the invention
The purpose of this invention is to provide a kind of method of from honey, extracting gene.
Technical scheme of the present invention adopts: basis of honey because of process for extracting, order is carried out according to the following steps:
(1) gets the above honey of 10 mL, add isopyknic pH and be 7.0 ~ 8.0 phosphate buffer soln, vibrated 1 ~ 3 hour; Fully centrifugal behind the mixing, abandoning supernatant adds 3 mL pH and is 7.0 ~ 8.0 phosphate buffer soln dissolution precipitation; Centrifugal, abandoning supernatant keeps throw out;
(2) add 1 mL lysis buffer in throw out, fully suspend, mixing, adding 10 μ L concentration is the Proteinase K solution of 20mg/mL, mixing is put 65 ℃ of heating in water bath 45 ~ 60 min, shakes frequently; Described lysis buffer is the mixed aqueous solution of cetyl trimethylammonium bromide, Tutofusin tris, YD 30 and sodium-chlor; Wherein the concentration of cetyl trimethylammonium bromide is 50mmol/L; The concentration of Tutofusin tris is 10mmol/L; The concentration of YD 30 is 2mmol/L, and the concentration of sodium-chlor is 400mmol/L, transfers pH to 8.0 with hydrochloric acid;
(3) take out, add 300 μ l saturated nacl aqueous solutions, thermal agitation 2 minutes places 0 ℃ of held 3 ~ 5 min, and is centrifugal;
(4) get supernatant, add isopyknic phenol: chloroform: the primary isoamyl alcohol volume ratio is the mixing solutions of 25:24:1, and mixing is centrifugal;
(5) get supernatant, add isopyknic chloroform: the primary isoamyl alcohol volume ratio is the mixing solutions of 24:1, and mixing is centrifugal;
(6) get supernatant, add 3 mol/L sodium acetate solns of 1/10th volumes, mixing adds and the isopyknic Virahol of mixed solution, and mixing was placed 2 hours at-20 ℃, and is centrifugal, and abandoning supernatant keeps throw out;
(7) taking precipitate with 75 ~ 80% washing with alcohol after, under the room temperature ethanol is dried up, at room temperature dry the honey DNA dry product of deposition maybe is dissolved in deposition in the TE solution of pH8.0, promptly get honey DNA reagent.The concentration of Tutofusin tris is 10mmol/L in the TE solution, and the concentration of YD 30 is 1mmol/L, and all the other are water.
In the inventive method, the described centrifugally operated of each step is the routine operation of this area DNA isolation, and centrifugal speed and time are generally centrifugal 10 min of 12 000 rpm, and operation at low temperatures as far as possible.
In the inventive method, described honey is nectar, secretory product or the honeydew of honeybee herborization, after self secretory product combines, through fully brewageing the crude sweet material that forms, like acacia honey, lichee honey etc.;
The inventive method has adopted the above honey of 10 mL to extract system; Improve the concentration of the honey DNA that obtains through the method that increases sample size; Adopt phosphate buffer soln to remove the soluble polysaccharide in the honey again; Utilize lysis buffer to make the plant constituent lysis in the honey; Sodium chloride solution with high density in lysing cell makes albumen precipitation, and utilizes CTAB under high ionic strength, can make polysaccharide precipitation but can not make the sedimentary principle of nucleic acid, effectively reduces the interference of gp to the DNA purifying.Further remove Deproteinization with phenol, chloroform, primary isoamyl alcohol mixing solutions, under coldcondition, make the nucleic acid deposition with Virahol at last, obtain complete segment, the highly purified honey DNA of high density.Gained honey DNA can satisfy the requirement of PCR reaction, makes to utilize molecular biology method to identify that honey quality is achieved.The inventive method result is stable, and favorable reproducibility is easy and simple to handle, need not specific apparatus and reagent.
Through measuring, the concentration of the honey DNA that the inventive method is extracted more than 100ng/ μ l, OD
260/ OD
280Ratio between 1.7 ~ 2.0, concrete data are referring to table 1 among the embodiment.The present invention adopts methods such as pcr amplification reaction and DNA agarose gel electrophoresis that different methods is extracted the honey DNA quality that obtains and detects.Detected result shows that the honey DNA quality that the inventive method is extracted is better, can obtain target P CR product when carrying out the PCR reaction, and the present invention can be widely used in the molecular Biological Detection field of honey.
The present invention is directed to the singularity of honey sample, developed honey DNA extraction method efficiently, successfully from polytype honey sample, extract and obtained high-quality honey DNA.On the basis of present method, can further carry out PCR reaction amplification, according to the plant origin of detected result honey sample that Rapid identification is extracted according to honey nectariferous plant kind designs specificity, narrow spectrum primer sequence.Like the honey outer packing mark is Japanese Honeysuckle honey; Extracting the honey sample DNA performing PCR of going forward side by side through the present invention detects; Reach PCR like the honey DNA that obtains and detect requirement, and can not augmentation detection go out the Japanese Honeysuckle special gene sequence, explain that then this honey is not to derive from Japanese Honeysuckle.The inventive method can be developed specific basis of honey because of extracting test kit, and the honey DNA that is extracted is the key reagents that is applied in the follow-up PCR detection, makes the applied molecular biology means identify that the plant origin of honey becomes possibility.
The present invention set up stable basis of honey because of process for extracting, and through many-sided experimental verification the safety and the validity of inventive method.The present invention can be widely used in basis of honey because of evaluation and the exploitation basis of honey of extraction, honey nectariferous plant kind because of extracting aspect such as test kit, have good economy and society benefit.
Description of drawings
Fig. 1 is a honey DNA electrophorogram, wherein swimming lane M:Marker DNA size criteria; P: corn gene group DNA reference substance; 1: the acacia honey DNA that high salt low pH method extracts among the embodiment 1; 2: the acacia honey DNA that phynol method extracts among the embodiment 2; 3: the acacia honey DNA that conventional CTAB method is extracted among the embodiment 3; 4-7: the honey DNA that embodiment 4-7 the inventive method is extracted;
Fig. 2 is the PCR product electrophorogram of the honey DNA that extracted of embodiment 1-7, wherein swimming lane M:Marker DNA size criteria; P: positive reference substance; The 1-7:PCR product; 8: blank.
Further set forth technical scheme of the present invention below in conjunction with accompanying drawing and embodiment, but method of the present invention application is not limited only to following examples.
Embodiment
Example 1 high salt low pH method extracts acacia honey DNA
1, material: acacia honey.
2, method:
Get 10mL honey, add the 10mL phosphate buffer soln, mixing.Vibration is 2 hours on the shaking table, centrifugal 10 min of 12 000 rpm.Abandoning supernatant adds 3mL phosphate buffer soln dissolution precipitation.Centrifugal 10 min of 12 000 rpm, abandoning supernatant adds 1mL lysis buffer dissolution precipitation.Adding 10 μ L concentration then is the Proteinase K solution of 20mg/mL, mixing, 65 ℃ of water-bath 45 min, centrifugal 10 min of 12 000 rpm.Get supernatant, add the liquor kalii acetici that isopyknic concentration is 2.5mmol/L, mixing, 0 ℃ of held 5 min precipitating proteins, centrifugal 10 min of 12 000 rpm.Get supernatant, add the sodium acetate soln of the 3mol/L of 1/10th volumes, mixing.Add and the isopyknic Virahol of mixed solution, mixing was placed 2 hours for-20 ℃.Take out centrifugal 10 min of 12 000 rpm, abandoning supernatant.Taking precipitate dries up ethanol under the room temperature after with 75% washing with alcohol, and the DNA deposition is dissolved in TE solution, obtains DNA reagent.
3, result: through the nucleic acid-protein analysis-e/or determining, adopt concentration and the purity of the acacia honey DNA that high salt low pH method extracted lower, the concentration of DNA is 13ng/ μ l, OD
260/ OD
280Ratio be 1.25.Get 5 μ L dna solutions in containing bromination electrophoresis detection in painted 0.8% sepharose of ingot, detected result is as shown in Figure 1, and swimming lane 1 shows there is not the DNA band, explains that high salt low pH method is not suitable for the extraction of honey DNA.
4, the extraction quality of PCR reaction detection DNA, method is following:
PCR reaction buffer system adopts premix, contains Taq enzyme 1.25U/25 μ L, each 0.4mmol/L of dNTP, Mg
2+Content is 3mmol/L.The PCR reaction system is 25 μ L, each 3 μ mol/L of primer concentration, and template consumption 1 μ L, pure water is supplied volume.Reaction parameter: behind 94 ℃ of preparatory sex change 5min, by 94 ℃ of sex change 30sec, 56 ℃ of annealing 30sec, 72 ℃ of extension 30sec programs are carried out 40 circulations, and last circulates back 72 ℃ and extends 10min, finishes reaction in 4 ℃.Forward primer is P1, and sequence is: CGAAATCGGTAGACGCTACG, reverse primer are P2, and sequence is: TTCCATTGAGTCTCTGCACCT.Get PCR product 5 μ L in containing bromination electrophoresis detection in painted 2% sepharose of ingot.
PCR product electrophorogram is seen Fig. 2, and swimming lane 1 shows there is not amplified band, and the honey DNA that the detected result explanation is extracted with high salt low pH method carries out the PCR reaction, can not obtain target P CR product.
1, material: acacia honey.
2, method:
Get 10mL honey, add the 10mL phosphate buffer soln, mixing.Centrifuge tube is placed on the shaking table vibration 2 hours, centrifugal 10 min of 12 000 rpm.Abandoning supernatant adds 3mL phosphate buffer soln dissolution precipitation, centrifugal 10 min of 12 000 rpm.Abandoning supernatant keeps throw out, adds 1mL lysis buffer dissolution precipitation.Adding 10 μ L concentration then is the Proteinase K solution of 20mg/mL, mixing, 65 ℃ of water-bath 45 min, centrifugal 10 min of 12 000 rpm.Get supernatant, add isopyknic phenol: chloroform: the primary isoamyl alcohol volume ratio is the mixing solutions of 25:24:1, mixing, centrifugal 10 min of 12 000 rpm.Get supernatant, add isopyknic chloroform: the primary isoamyl alcohol volume ratio is the mixing solutions of 24:1, centrifugal 10 min of 12 000 rpm.Get supernatant, add isopyknic chloroform: the primary isoamyl alcohol volume ratio is the mixing solutions of 24:1, centrifugal 10 min of 12 000 rpm.Get supernatant, add the sodium acetate soln of 3 mol/L of 1/10th volumes, mixing adds and the isopyknic Virahol of mixed solution then, and mixing was placed 2 hours for-20 ℃.Take out centrifugal 10 min of 12 000 rpm, abandoning supernatant.Taking precipitate dries up ethanol under the room temperature after with 75% washing with alcohol, and the DNA deposition is dissolved in TE solution, obtains DNA reagent.
3, result: through the nucleic acid-protein analysis-e/or determining, after the repeatedly extracting of sample through phenol and chloroformic solution, the concentration of gained honey DNA is 11ng/ μ l, OD
260/ OD
280Ratio be 1.43.Get 5 μ L dna solutions in containing bromination electrophoresis in painted 0.8% sepharose of ingot, detected result is as shown in Figure 1, and swimming lane 2 shows there is not the DNA band, explains that phynol method is not suitable for the extraction of honey DNA.
4, the extraction quality of PCR reaction detection DNA, method be with embodiment 1,
PCR product electrophorogram is seen Fig. 2, and swimming lane 2 shows there is not amplified band, and the honey DNA that the detected result explanation is extracted with phynol method carries out the PCR reaction, can not obtain target P CR product.
1, material: acacia honey.
2, method:
Get 10mL honey, add the 10mL phosphate buffer soln, mixing.Centrifuge tube is placed on the shaking table vibration 2 hours, centrifugal 10 min of 12 000 rpm.Abandoning supernatant adds 3mL phosphate buffer soln dissolution precipitation, centrifugal 10 min of 12 000 rpm.Abandoning supernatant keeps throw out, adds 1mL lysis buffer dissolution precipitation.Adding 10 μ L concentration then is the Proteinase K solution of 20mg/mL, mixing, 65 ℃ of water-bath 45 min, centrifugal 10 min of 12 000 rpm.Get supernatant, add isopyknic chloroform: the primary isoamyl alcohol volume ratio is the mixing solutions of 24:1, centrifugal 10 min of 12 000 rpm.Get supernatant, add the sodium acetate soln of 3 mol/L of 1/10th volumes, mixing adds and the isopyknic Virahol of mixed solution then, and mixing was placed 2 hours for-20 ℃.Take out centrifugal 10 min of 12 000 rpm, abandoning supernatant.Taking precipitate dries up ethanol under the room temperature after with 75% washing with alcohol, and the DNA deposition is dissolved in TE solution, obtains DNA reagent.
3, result: through the nucleic acid-protein analysis-e/or determining, the concentration of the honey DNA that conventional CTAB method is extracted is 16ng/ μ l, OD
260/ OD
280Ratio be 1.39.Get 5 μ L dna solutions in containing bromination electrophoresis in painted 0.8% sepharose of ingot, detected result is as shown in Figure 1, and swimming lane 3 shows there is not the DNA band, explains that conventional CTAB method is not suitable for the extraction of honey DNA.
4, the extraction quality of PCR reaction detection DNA, method be with embodiment 1,
PCR product electrophorogram is seen Fig. 2, and swimming lane 3 shows there is not amplified band, and the honey DNA that the electrophoresis result explanation is extracted with conventional CTAB method carries out the PCR reaction, can not obtain target P CR product.
1, material: acacia honey.
2, method:
The preparation of reagent
PH is 7.0 ~ 8.0 phosphate buffer soln: the phosphate buffer soln compound method is seen document: " molecular cloning experiment guide ", J. Sa nurse Brooker D.W. La Saier work, Huang Peitang etc. translate, Science Press, 1, the 1570 page of appendix.Concrete operations: take by weighing 8.00 gram sodium-chlor, 0.20 gram Repone K, 1.44 gram Sodium phosphate, dibasics, 0.24 gram potassium primary phosphate, after the suitable quantity of water dissolving, pH value to 7 ~ 8 with hydrochloric acid conditioning solution are settled to 1L, and high pressure steam sterilization after the packing is stored in room temperature.
Lysis buffer: take by weighing 18.20 gram cetyl trimethylammonium bromides, 23.38 gram sodium-chlor, 1.21 gram Tutofusin triss; 0.60 gram YD 30; After the suitable quantity of water dissolving, the pH value to 8.0 with hydrochloric acid conditioning solution is settled to 1L; High pressure steam sterilization after the packing is stored in room temperature.
TE solution: take by weighing 1.21 gram Tutofusin triss, 0.30 gram YD 30, after the suitable quantity of water dissolving, the pH value to 8.0 with hydrochloric acid conditioning solution is settled to 1L, and high pressure steam sterilization after the packing is stored in room temperature.
The extraction of DNA: 10 mL honey are got in (1), add 10 mL phosphate buffer solns, and vibration is 2 hours on the shaking table, fully mixing; Centrifugal 10 min of 12 000 rpm, abandoning supernatant adds 3 mL phosphate buffer soln dissolution precipitations; Centrifugal, abandoning supernatant keeps throw out; (2) add 1 mL lysis buffer in throw out, fully suspend, adding 10 μ L concentration is the Proteinase K solution of 20 mg/mL, and mixing is put 65 ℃ of heating in water bath 45 min, shakes frequently; (3) take out, add 300 μ l saturated nacl aqueous solutions, thermal agitation 2 minutes places 0 ℃ of held 5 min, and is centrifugal; (4) get supernatant, add isopyknic phenol: chloroform: the primary isoamyl alcohol volume ratio is the mixing solutions of 25:24:1, and mixing is centrifugal; (5) get supernatant, add isopyknic chloroform: the primary isoamyl alcohol volume ratio is the mixing solutions of 24:1, and mixing is centrifugal; (6) get supernatant, add 3 mol/L sodium acetate solns of 1/10th volumes, mixing adds and the isopyknic Virahol of mixed solution, and mixing was placed 2 hours at-20 ℃, and is centrifugal, and abandoning supernatant keeps throw out; (7) taking precipitate with 75% washing with alcohol after, under the room temperature ethanol is dried up, deposition is dissolved in the TE solution of pH8.0, obtain honey DNA reagent.
3, result: through the nucleic acid-protein analysis-e/or determining, the concentration of the acacia honey DNA that the inventive method is extracted is 108ng/ μ l, OD
260/ OD
280Ratio be 1.82.Get 5 μ L dna solutions in containing bromination electrophoresis in painted 0.8% sepharose of ingot, result such as Fig. 1, swimming lane 4 show sample DNA bands are clear, explain that the DNA quality that the inventive method extracts is better.
4, the extraction quality of PCR reaction detection DNA, method be with embodiment 1,
PCR product electrophorogram is seen Fig. 2, and swimming lane 4 shows that the DNA that the inventive method is extracted can obtain the PCR product, and the products therefrom size meets experimental design, and the amplified fragments size is 180bp.
1, material: Motherwort Herb honey.
2, method is with embodiment 4:
Get 15mL honey, add the 15mL phosphate buffer soln, handle, the at room temperature dry at last honey DNA dry product that gets by the extraction step of embodiment 4.
3, result: use TE solution dissolving DNA dry product, through the nucleic acid-protein analysis-e/or determining, the concentration of the Motherwort Herb honey DNA that the inventive method is extracted is 115ng/ μ l, OD
260/ OD
280Ratio be 1.85.Get 5 μ L dna solutions in containing bromination electrophoresis in painted 0.8% sepharose of ingot, result such as Fig. 1, swimming lane 5 show sample DNA bands are clear, explain that the DNA quality that the inventive method extracts is better.
4, the extraction quality of PCR reaction detection DNA, method are with embodiment 1:
PCR product electrophorogram is seen Fig. 2, and swimming lane 5 shows that the DNA that the inventive method is extracted can obtain the PCR product, and the products therefrom size meets experimental design, and the amplified fragments size is 180bp.
1, material: sweet-scented osmanthus honey.
2, method is with embodiment 4.
3, result: through the nucleic acid-protein analysis-e/or determining, the concentration of the sweet-scented osmanthus honey DNA that the inventive method is extracted is 137ng/ μ l, OD
260/ OD
280Ratio be 1.78.Get 5 μ L dna solutions in containing bromination electrophoresis in painted 0.8% sepharose of ingot, result such as Fig. 1, swimming lane 6 show sample DNA bands are clear, explain that the honey DNA quality that the inventive method extracts is better.
4, the extraction quality of PCR reaction detection DNA, method are with embodiment 1:
PCR product electrophorogram is seen Fig. 2, and swimming lane 6 shows that the DNA that the inventive method is extracted can obtain the PCR product, and the products therefrom size meets experimental design, and the amplified fragments size is 180bp.
1, material: lichee honey.
2, method is with embodiment 4.
3, result: through the nucleic acid-protein analysis-e/or determining, the concentration of the lichee honey DNA that the inventive method is extracted is 106ng/ μ l, OD
260/ OD
280Ratio be 1.89.Get 5 μ L dna solutions in containing bromination electrophoresis in painted 0.8% sepharose of ingot, result such as Fig. 1, swimming lane 7 show sample DNA bands are clear, explain that the DNA quality that the inventive method extracts is better.
4, the extraction quality of PCR reaction detection DNA, method are with embodiment 1:
PCR product electrophorogram is seen Fig. 2, and swimming lane 7 shows that the DNA that the inventive method is extracted can obtain the PCR product, and the products therefrom size meets experimental design, and the amplified fragments size is 180bp.
Concentration and the purity result of the honey DNA of 7 embodiment extractions see the following form 1.
Concentration and the purity of the honey DNA that table 1 different methods extracts
Concentration and the purity of the honey DNA that different methods extracted that subordinate list 1 records for the nucleic acid-protein analyser are relatively found, adopt concentration and the purity of the acacia honey DNA that high salt low pH method extracted lower, and DNA concentration is 13ng/ μ L, OD
260/ OD
280Be 1.25.The acacia honey DNA concentration of taking phynol method to extract is lower, and purity does not reach the requirement of follow-up PCR reaction yet, and DNA concentration is 11ng/ μ L, OD
260/ OD
280Be 1.43.Adopt the concentration of the acacia honey DNA that conventional CTAB method extracted and the requirement that purity does not reach the PCR reaction yet, DNA concentration is 16ng/ μ L, OD
260/ OD
280Be 1.39.It is higher that the inventive method is extracted the honey DNA quality that obtains, the concentration of DNA more than 100ng/ μ l, OD
260/ OD
280Ratio is between 1.7 ~ 2.0.
Claims (1)
- A basis of honey because of process for extracting, it is characterized in that order is carried out according to the following steps:(1) gets the above honey of 10 mL, add isopyknic pH and be 7.0 ~ 8.0 phosphate buffer soln, vibrated 1 ~ 3 hour; Fully centrifugal behind the mixing, abandoning supernatant adds 3 mL pH and is 7.0 ~ 8.0 phosphate buffer soln dissolution precipitation; Centrifugal, abandoning supernatant keeps throw out;(2) add 1 mL lysis buffer in throw out, fully suspend, mixing, adding 10 μ L concentration is the Proteinase K solution of 20mg/mL, mixing is put 65 ℃ of heating in water bath 45 ~ 60 min, shakes frequently; Described lysis buffer is the mixed aqueous solution of cetyl trimethylammonium bromide, Tutofusin tris, YD 30 and sodium-chlor; Wherein the concentration of cetyl trimethylammonium bromide is 50mmol/L; The concentration of Tutofusin tris is 10mmol/L; The concentration of YD 30 is 2mmol/L, and the concentration of sodium-chlor is 400mmol/L, transfers pH to 8.0 with hydrochloric acid;(3) take out, add 300 μ l saturated nacl aqueous solutions, thermal agitation 2 minutes places 0 ℃ of held 3 ~ 5 min, and is centrifugal;(4) get supernatant, add isopyknic phenol: chloroform: the primary isoamyl alcohol volume ratio is the mixing solutions of 25:24:1, and mixing is centrifugal;(5) get supernatant, add isopyknic chloroform: the primary isoamyl alcohol volume ratio is the mixing solutions of 24:1, and mixing is centrifugal;(6) get supernatant, add 3 mol/L sodium acetate solns of 1/10th volumes, mixing adds and the isopyknic Virahol of mixed solution, and mixing was placed 2 hours at-20 ℃, and is centrifugal, and abandoning supernatant keeps throw out;(7) taking precipitate with 75 ~ 80% washing with alcohol after, under the room temperature ethanol is dried up, at room temperature dry the honey DNA dry product of deposition maybe is dissolved in deposition in the TE solution of pH8.0, promptly get honey DNA reagent; The concentration of Tutofusin tris is 10mmol/L in the said TE solution, and the concentration of YD 30 is 1mmol/L, and all the other are water.
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| CN103509786A (en) * | 2013-05-21 | 2014-01-15 | 西北农林科技大学 | Extraction method and applications of inset dry specimen genome |
| CN111434243A (en) * | 2019-01-23 | 2020-07-21 | 重庆悬泉蜂业有限公司 | Deep processing extraction method for extracting active substances from honey |
| KR20220026093A (en) * | 2020-08-25 | 2022-03-04 | 경기대학교 산학협력단 | Method of DNA isolation using affinity column in sugar honey |
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| CN103215373A (en) * | 2013-05-10 | 2013-07-24 | 中国检验检疫科学研究院 | Honey detection composition, kit, detection method and application |
| CN103215373B (en) * | 2013-05-10 | 2014-11-05 | 中国检验检疫科学研究院 | Honey detection composition, kit, detection method and application |
| CN103509786A (en) * | 2013-05-21 | 2014-01-15 | 西北农林科技大学 | Extraction method and applications of inset dry specimen genome |
| CN111434243A (en) * | 2019-01-23 | 2020-07-21 | 重庆悬泉蜂业有限公司 | Deep processing extraction method for extracting active substances from honey |
| KR20220026093A (en) * | 2020-08-25 | 2022-03-04 | 경기대학교 산학협력단 | Method of DNA isolation using affinity column in sugar honey |
| KR102371553B1 (en) | 2020-08-25 | 2022-03-07 | 경기대학교 산학협력단 | Method of DNA isolation using affinity column in sugar honey |
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