[go: up one dir, main page]

CN106754899A - A kind of method for extracting tan leather DNA - Google Patents

A kind of method for extracting tan leather DNA Download PDF

Info

Publication number
CN106754899A
CN106754899A CN201710183413.1A CN201710183413A CN106754899A CN 106754899 A CN106754899 A CN 106754899A CN 201710183413 A CN201710183413 A CN 201710183413A CN 106754899 A CN106754899 A CN 106754899A
Authority
CN
China
Prior art keywords
centrifuge tube
leather
centrifugation
volume
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710183413.1A
Other languages
Chinese (zh)
Inventor
杨芳芳
罗利玲
张建扬
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Fiber Product Detects Research Institute
Original Assignee
Guangzhou Fiber Product Detects Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Fiber Product Detects Research Institute filed Critical Guangzhou Fiber Product Detects Research Institute
Priority to CN201710183413.1A priority Critical patent/CN106754899A/en
Publication of CN106754899A publication Critical patent/CN106754899A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Landscapes

  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The method that the present invention extracts tan leather DNA is comprised the following steps:S2, in the first centrifuge tube add leather lysis buffer and Proteinase K;Add leather sample;S3, incubation;S4, lysate is suctioned out from the first centrifuge tube to the second centrifuge tube;S5, to the second centrifuge tube add tris saturated phenols, mix centrifugation;S6, supernatant liquid is inhaled from the second centrifuge tube to the 3rd centrifuge tube, add tris saturated phenols, mix centrifugation;S7, supernatant liquid is inhaled from the 3rd centrifuge tube to the 4th centrifuge tube, add chloroform, mix centrifugation;S8, supernatant liquid is inhaled from the 4th centrifuge tube to the 5th centrifuge tube, add sodium acetate solution and precooling absolute ethyl alcohol, be centrifuged after standing;The supernatant of the 5th centrifuge tube that S9, suction are abandoned after centrifugation, adds ethanol, and liquid is transferred into the 6th centrifuge tube, is centrifuged, and supernatant is abandoned in suction;S10, the residue in the 6th centrifuge tube is dried, adds the DNA on TE buffer solution tube walls;S11, the DNA concentration for determining extraction.The present invention can rapid extraction leather DNA.

Description

A kind of method for extracting tan leather DNA
【Technical field】
The biogenic technical field that technique of gene detection differentiates leather and leather and fur products is the present invention relates to the use of, specifically It is to be related to a kind of method for extracting tan leather DNA.
【Background technology】
The method standard for never being differentiated for leather and fur products animal species both at home and abroad.For leather and fur products, mesh Before the standard that can refer to be ISO 17131:2012《Leather-leather differentiates microscopic method》And QB/T2262-1996《Leather industry Term》, wherein ISO 17131:2012 are only applicable to distinguish leather and non-leather, and QB/T 2262-1996 only define leather work Industry term, specific discrimination method is not mentioned.The missing of examination criteria, causes substantial amounts of leather and fur products just to be flowed without inspection Enter market.
At present, the discrimination method of leather and fur goods animal species is actively studied both at home and abroad, mainly there are 4 classes basic Method:
1) sense organ experience differential method:Current domestic each testing agency's Main Basiss sense organ experience, i.e., soon, hand touch, nose The means such as news, the animal species to leather and fur goods are observed.But, sense organ experience differential method is to the special of reviewer Industry knowledge and detection experience requirement are higher, and are the results of subjective judgement, so the examining report between each testing agency goes out often Existing inconsistent phenomenon.In addition, with the progress of modern processing, people can change the pore of product according to customer requirement Arrangement and surface texture, this proposes challenge to traditional discrimination method.Additionally, specialty is cracked down on counterfeit goods, personage also often holds detection machine The different examining reports that structure is provided, unwarranted demand, the normal operation of very disruptive testing agency and enterprise are proposed to enterprise.
2) microscope differential method:Microscopic method is mainly by light microscope or electron microscope to leather and fur system The fibre morphology structure of product is observed, but microscope differential method is applied to the larger leather of Morphological Differences and fur system Product, for the less sample of Morphological Differences, microscopic method is also not easy to distinguish.
3) infrared spectrum differential method:Infrared spectrum differential method is the result with Substance Interactions based on infrared light, i.e., With the Infrared irradiation sample of continuous wavelength, when the energy of light is exactly equal to the difference of the energy of ground state and a certain excitation state, point Son can absorb the energy of infrared light, make molecule, the vibration of atom and rotational energy level that transition to occur, so as to produce INFRARED ABSORPTION light Spectrum.But, leather and fur products generally goes through the processing technology of complexity, and its surface often contains coatings, infrared spectrum can be produced Considerable influence, so as to increase the difficulty of discriminating.So the research and application of infra-red sepectrometry discriminating leather and fur goods are current Also in the exploratory stage.
4) molecular biotechnology differential method:Molecular biotechnology primarily directed to DNA analysis method, using showing biological characteristic The different DNA sequence dna information that have of various living species differentiated that it can be broken through according to animal origin morphosis The limitation of discriminating, compared with traditional analysis, more with objectivity and accuracy.DNA is by the acid of deoxyribose core former times The long-chain polymer of composition, the molecular size (molecular weight or length) of different biology DNA, structure have certain difference.Therefore, Being detected by DNA can relatively accurately differentiate various animal origins and leather.Conventional molecular biotechnology method includes PCR Technology, genetic barcode technology and two generation high-flux sequence analytical technologies.But use this discrimination method, the extraction ratio of DNA Cumbersome, the consuming time is long, high cost.
Therefore, a kind of method that can quickly, easily extract tan leather DNA is needed badly.
【The content of the invention】
It is an object of the invention to overcome the shortcomings of above-mentioned technology, there is provided a kind of method of extraction tan leather DNA.
A kind of method of extraction tan leather DNA that the present invention is provided, including the following steps for sequentially carrying out:
S2, to adding the leather lysis buffer and Proteinase K that prepare in the first centrifuge tube, leather lysis buffer and The volume ratio of Proteinase K is 975:25;To leather sample is added in first centrifuge tube, adding the amount of leather sample should not make Outside liquid discharger;
S3, the lid for covering first centrifuge tube, after concussion is mixed, 53 to 58 DEG C of gold are placed in by first centrifuge tube Category bath is incubated 40-48 hours;
In suctioning out lysate to second centrifuge tube of the first volume in S4, first centrifuge tube from after incubation;
S5, to the tris saturated phenols that the first volume is added in second centrifuge tube, overturn mix, to be not less than 12000 Rev/min centrifugation more than 20 minutes;
In S6, second centrifuge tube from after centrifugation in suction supernatant liquid to the 3rd centrifuge tube, the supernatant liquid inhaled Volume be the second volume;To the tris saturated phenols that the second volume is added in the 3rd centrifuge tube, overturn and mix, to be not less than 12000 revs/min of centrifugation more than 20 minutes;
In S7, the 3rd centrifuge tube from after centrifugation in suction supernatant liquid to the 4th centrifuge tube, the supernatant liquid inhaled Volume be third volume;And to the chloroform of third volume is added in the 4th centrifuge tube, overturn and mix, to be not less than 12000 revs/min of centrifugation more than 20 minutes;
In S8, the 4th centrifuge tube from after centrifugation in suction supernatant liquid to the 5th centrifuge tube, the supernatant liquid inhaled Volume be fourth volume;To the sodium acetate solution of 1/10 times of fourth volume of addition in the 5th centrifuge tube and 2.5 times the 4th The precooling absolute ethyl alcohol of volume, more than 2 hours are stood after mixing, then being not less than 13000 revs/min of 25 points of centrifugation It is more than clock;
After supernatant liquor in the 5th centrifuge tube that S9, suction are abandoned after centrifugation, to adding the in the 5th centrifuge tube 75% ethanol of five volumes, with pipettor purge tube wall, then liquid is transferred in the 6th centrifuge tube, and to the described 6th from Heart pipe is upper in the 6th centrifuge tube that suction is abandoned after centrifugation to be not less than 13000 revs/min of centrifugation more than 10 minutes Layer clear liquid;
S10, the residue in the 6th centrifuge tube is dried, until alcohol-free residual, is subsequently adding hexasomic DNA on long-pending TE buffer solution tube walls;
S11, the DNA concentration for determining extraction.
Further, step S1 is also included before step S2:A block leather sample is cut, the coatings of leather sample are scraped Go, shred leather sample.
Further, in step S2, the preparation of the leather lysis buffer is comprised the following steps:
By the Tris-Cl buffer solutions of 20mmol/L, the EDTA disodium ethylene diamine tetraacetates of 5mmol/L, 400mmol/L NaCl sodium chloride, the SDS dodecyl sodium sulfates of 1% (m/v) and 1% CTAB cetyl trimethylammonium bromides are matched somebody with somebody in proportion It is good;
Solution after to preparing carries out filtration sterilization.
Further, filtration sterilization is carried out with the nitrocellulose filter of 0.22um to the solution for preparing.
Further, in step S10, Tris-Cl buffer solution and 1mmol/L of the TE buffer solutions including 10mmol/L EDTA disodium ethylene diamine tetraacetates.
Further, the pH value of the Tris-Cl buffer solutions and EDTA disodium ethylene diamine tetraacetates is all 8.0.
Further, in step S10, the mode being dried to the residue in the 6th centrifuge tube is to dry or put It is dried in 50 DEG C of temperature.
Further, in step S3, first centrifuge tube is placed in 56 DEG C of metal baths and is incubated 40-48 hours.
Further, in step S2, the volume of the leather lysis buffer is 975ul, and the volume of the Proteinase K is 25ul, the weight of the leather sample of addition is 0.1g;In step S4, first volume is 2ml;In step S8, the acetic acid Sodium solution is 3mol/L sodium acetate solutions, and the precooling absolute ethyl alcohol is -20 DEG C of precooling absolute ethyl alcohols, is mixed after in -20 DEG C Stand 3 hours;In step S9, the 5th volume is 1ml;In step S10, the hexasomic product is 50ul.
Further, the centrifugal speed in step S5, in step S6 neutralization procedures S7 all for 12000 revs/min, centrifugation when Between all be 25 minutes;Centrifugal speed in step S8 is 13000 revs/min, and centrifugation time is 30 minutes;Centrifugation in step S9 Speed is 13000 revs/min, and centrifugation time is 15 minutes.
Implement the present invention, can quickly, simply, efficiently extract the DNA of tan leather or leather and fur products, extract DNA consumptions The time taken is shorter, low cost, can be to differentiate that leather and leather and fur products provide high-quality DNA samples using molecular biotechnology Product so that the discriminating of the animal species of leather or leather and fur products is accurate and efficient.
【Brief description of the drawings】
A kind of flow chart of the method for extraction tan leather DNA that Fig. 1 is provided for the present invention.
【Specific embodiment】
The invention will be further described with reference to the accompanying drawings and examples.
With reference to Fig. 1, a kind of method for extracting tan leather DNA (DNA) that the present invention is provided, can be use Molecular biotechnology discriminating leather and leather and fur products provide high-quality sample DNA so that detection is accurate and efficient.The method bag Include the following steps for sequentially carrying out:
S1, a block leather sample is cut, the length of side of leather sample is about 5 centimetres.The coatings of leather sample are scraped off, with Anti- coatings influence the measure of DNA concentration.Shred leather sample.
The leather lysis buffer and Proteinase K for preparing, leather are added in S2, the first centrifuge tube to 1ml (milliliter) The volume ratio of lysis buffer and Proteinase K is 975:25.In the present embodiment, what leather lysis buffer and Proteinase K were added Amount is respectively 975ul (microlitre) and 25ul (microlitre).Then with aseptic nipper grip leather sample add pipe in, leather sample Weight is 0.1g.The amount of leather sample can be weighed by electronic balance.Adding the amount of leather sample should not make outside liquid discharger.
S3, the lid for covering the first centrifuge tube, after concussion is mixed, are placed in the first centrifuge tube 53 to 58 DEG C of metal baths and are incubated 40-48 hours, cracked, made DNA separate outs from nucleoprotein, so as to obtain the lysate that DNA, protein are separated. In the present embodiment, the first centrifuge tube is placed in preferably 56 DEG C metal baths and is incubated.
In S4, the first centrifuge tube from after incubation by pipettor suction out the first volume lysate to 15ml second from In heart pipe.In the present embodiment, the first volume is 2ml.
S5, to tris (trishydroxymethylaminomethane) saturated phenol that the first volume is added in the second centrifuge tube, for removing Indigested protein in liquid.It is reverse to mix, it is careful not to firmly shake, by centrifuge being not less than 12000 revs/min Centrifugation more than 20 minutes, DNA is present in the water phase on upper strata in the liquid for obtaining.Preferably, the speed of centrifugation is 12000 revs/min, centrifugation time is 25 minutes.
In S6, the second centrifuge tube from after centrifugation by pipettor inhale supernatant liquid to the 3rd centrifuge tube of 15ml in, institute The volume of the supernatant liquid of suction is the second volume.To the tris saturated phenols that the second volume is added in the 3rd centrifuge tube, with further Remove indigested protein in liquid.It is reverse to mix, by centrifuge being not less than 12000 revs/min of centrifugation 20 More than minute.Preferably, the speed of centrifugation is 12000 revs/min, and centrifugation time is 25 minutes.
In S7, the 3rd centrifuge tube from after centrifugation by pipettor inhale supernatant liquid to the 4th centrifuge tube of 15ml in, institute The volume of the supernatant liquid of suction is third volume.To the chloroform that third volume is added in the 4th centrifuge tube, chloroform contributes to water phase With the phenol in organic phase separation and removing liquid.It is reverse to mix, by centrifuge being not less than 12000 revs/min of speed Centrifugation more than 20 minutes, obtains the DNA solution on upper strata.Preferably, the speed of centrifugation is 12000 revs/min, and centrifugation time is 25 Minute.
In S8, the 4th centrifuge tube from after centrifugation by pipettor inhale supernatant liquid to the 5th centrifuge tube of 15ml in, institute The volume of the supernatant liquid of suction is fourth volume.To added in the 5th centrifuge tube 1/10 times of sodium acetate solution of fourth volume and 2.5 times of precooling absolute ethyl alcohols of fourth volume, make to be precipitated under DNA, mix and stand more than 2 hours after -20 DEG C, and preferably 3 is small When.Then by centrifuge being not less than 13000 revs/min of centrifugation more than 25 minutes.Preferably, sodium acetate solution is 3mol/L (mole every liter) sodium acetate solution, precooling absolute ethyl alcohol is -20 DEG C of precooling absolute ethyl alcohols.The speed of centrifugation is 13000 Rev/min, centrifugation time is 30 minutes.
Supernatant in the 5th centrifuge tube that S9, suction are abandoned after centrifugation, to the 5th volume of addition in the 5th centrifuge tube 75% ethanol, for the salt in the DNA for washing away lower precipitation.Tube wall is purged with pipettor, then liquid is shifted by pipettor Into the 6th centrifuge tube of 1ml, and pass through centrifuge to the 6th centrifuge tube to be not less than 13000 revs/min of centrifugation 10 More than minute.Supernatant in the 6th centrifuge tube that suction is abandoned after centrifugation.In the present embodiment, the 5th volume is 1ml.The speed of centrifugation It is 13000 revs/min to spend, and centrifugation time is 15 minutes.
S10, it is that DNA is dried to the residue in the 6th centrifuge tube, until alcohol-free residual, is subsequently adding hexasomic DNA on long-pending TE buffer solution tube walls.In the present embodiment, hexasomic product is 50ul (microlitre).Wherein, to the 6th centrifuge tube The mode that interior DNA is dried is to dry.In another alternative, the 6th centrifuge tube can be placed in 50 DEG C of temperature Row drying.TE buffer solutions include that the Tris-Cl buffer solutions of 10mmol/L (mM every liter) (that is, are added suitable in Tris solution The hydrochloric acid of equivalent) and 1mmol/L EDTA (disodium ethylene diamine tetraacetate).Tris-Cl buffer solutions and EDTA (ethylenediamine tetra-acetic acids Disodium) pH value all be 8.0, be easy to make DNA be dissolved in water phase.
S11, the DNA concentration that extraction is determined by ELIASA, can so be identified using molecular biotechnology differential method Which kind of animal species leather sample belongs to.
The preparation of leather lysis buffer of the invention is comprised the following steps:
First, by the Tris-Cl buffer solutions of 20mmol/L, the EDTA (disodium ethylene diamine tetraacetate) of 5mmol/L, The NaCl (sodium chloride) of 400mmol/L, the SDS (dodecyl sodium sulfate) of 1% (m/v) and 1% CTAB (cetyl front threes Base ammonium bromide) prepare in proportion.The pH value of Tris-Cl buffer solutions and EDTA (disodium ethylene diamine tetraacetate) is all 8.0, is easy to DNA is dissolved in water phase.SDS (dodecyl sodium sulfate) and CTAB (cetyl trimethylammonium bromide) can be by leather samples Cell membrane lysis, digesting protein or polypeptide in the presence of Proteinase K and EDTA (disodium ethylene diamine tetraacetate), make nucleoprotein Denaturation degraded, so that DNA separate outs from nucleoprotein.
Then to preparing after solution carry out filtration sterilization with the nitrocellulose filter of 0.22um (micron).
Implement after the method for extraction DNA of the invention, tan leather or leather system can be extracted rapidly, high-efficient simple The DNA of product, it is short to expend the time, low cost, can be to differentiate that leather and leather and fur products provide high-quality using molecular biotechnology The sample of DNA, can so identify the animal species of leather or leather and fur products by molecular biotechnology differential method, differentiate accurate Really and efficiently.Be meet using molecular biotechnology differential method discriminating DNA concentration requirement, can be ready to many parts it is identical Leather sample DNA extractions are carried out by the method for the present invention simultaneously, the DNA concentration for so extracting is just more accurate, further Improve the degree of accuracy and efficiency for differentiating.
Above example only expresses the preferred embodiment of the present invention, and its description is more specific and detailed, but can not Therefore it is interpreted as the limitation to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, Without departing from the inventive concept of the premise, various modifications and improvements can be made, it is such as special to the difference in each embodiment Levy and be combined, these belong to protection scope of the present invention.

Claims (10)

1. a kind of method for extracting tan leather DNA, it is characterised in that:Including the following steps for sequentially carrying out:
S2, to adding the leather lysis buffer and Proteinase K that prepare, leather lysis buffer and albumen in the first centrifuge tube The volume ratio of enzyme K is 975:25;To leather sample is added in first centrifuge tube, adding the amount of leather sample should not make liquid Outside discharger;
S3, the lid for covering first centrifuge tube, after concussion is mixed, 53 to 58 DEG C of metal baths are placed in by first centrifuge tube It is incubated 40-48 hours;
In suctioning out lysate to second centrifuge tube of the first volume in S4, first centrifuge tube from after incubation;
S5, to the tris saturated phenols that the first volume is added in second centrifuge tube, overturn mix, to be not less than 12000 revs/min The centrifugation of clock more than 20 minutes;
In S6, second centrifuge tube from after centrifugation in suction supernatant liquid to the 3rd centrifuge tube, the body of the supernatant liquid inhaled Product is the second volume;To the tris saturated phenols that the second volume is added in the 3rd centrifuge tube, overturn and mix, to be not less than 12000 revs/min of centrifugation more than 20 minutes;
In S7, the 3rd centrifuge tube from after centrifugation in suction supernatant liquid to the 4th centrifuge tube, the body of the supernatant liquid inhaled Product is third volume;To the chloroform that third volume is added in the 4th centrifuge tube, overturn and mix, to be not less than 12000 revs/min The centrifugation of clock more than 20 minutes;
In S8, the 4th centrifuge tube from after centrifugation in suction supernatant liquid to the 5th centrifuge tube, the body of the supernatant liquid inhaled Product is fourth volume;To the sodium acetate solution and 2.5 times of fourth volumes of 1/10 times of fourth volume of addition in the 5th centrifuge tube Precooling absolute ethyl alcohol, stand more than 2 hours after mixing, then be not less than 13000 revs/min of centrifugation 25 minutes with On;
After supernatant liquor in the 5th centrifuge tube that S9, suction are abandoned after centrifugation, to adding five body constituents in the 5th centrifuge tube 75% long-pending ethanol, tube wall is purged with pipettor, and then liquid is transferred in the 6th centrifuge tube, and to the 6th centrifuge tube To be not less than 13000 revs/min of centrifugation more than 10 minutes, the upper strata in the 6th centrifuge tube that suction is abandoned after centrifugation is clear Liquid;
S10, the residue in the 6th centrifuge tube is dried, until alcohol-free residual, is subsequently adding hexasomic product DNA on TE buffer solution tube walls;
S11, the DNA concentration for determining extraction.
2. a kind of method for extracting tan leather DNA according to claim 1, it is characterised in that:Also wrapped before step S2 Include step S1:A block leather sample is cut, the coatings of leather sample are scraped off, shred leather sample.
3. a kind of method for extracting tan leather DNA according to claim 1, it is characterised in that:In step S2, the skin The preparation of lysis buffer is removed from office, is comprised the following steps:
By the Tris-Cl buffer solutions of 20mmol/L, the EDTA disodium ethylene diamine tetraacetates of 5mmol/L, 400mmol/L NaCl chlorine Change sodium, the SDS dodecyl sodium sulfates of 1% (m/v) and 1% CTAB cetyl trimethylammonium bromides to prepare in proportion;
Solution after to preparing carries out filtration sterilization.
4. a kind of method for extracting tan leather DNA according to claim 3, it is characterised in that:Solution to preparing is used The nitrocellulose filter of 0.22um carries out filtration sterilization.
5. a kind of method for extracting tan leather DNA according to claim 1, it is characterised in that:It is described in step S10 TE buffer solutions include the Tris-Cl buffer solutions of 10mmol/L and the EDTA disodium ethylene diamine tetraacetates of 1mmol/L.
6. the method for a kind of extraction tan leather DNA according to claim 3 or 5, it is characterised in that:The Tris-Cl The pH value of buffer solution and EDTA disodium ethylene diamine tetraacetates is all 8.0.
7. a kind of method for extracting tan leather DNA according to claim 1, it is characterised in that:In step S10, to institute The mode that the residue in the 6th centrifuge tube is dried is stated to be dried to dry or being placed in 50 DEG C of temperature.
8. a kind of method for extracting tan leather DNA according to claim 1, it is characterised in that:In step S3, will be described First centrifuge tube is placed in 56 DEG C of metal baths and is incubated 40-48 hours.
9. a kind of method for extracting tan leather DNA according to claim 1, it is characterised in that:In step S2, the skin The volume for removing from office lysis buffer is 975ul, and the volume of the Proteinase K is 25ul, and the weight of the leather sample of addition is 0.1g; In step S4, first volume is 2ml;In step S8, the sodium acetate solution is 3mol/L sodium acetate solutions, the precooling Absolute ethyl alcohol is -20 DEG C of precooling absolute ethyl alcohols, is mixed after standing 3 hours in -20 DEG C;In step S9, the 5th volume is 1ml;In step S10, the hexasomic product is 50ul.
10. a kind of method for extracting tan leather DNA according to claim 1, it is characterised in that:In step S5, step Centrifugal speed in S6 neutralization procedures S7 is all 12000 revs/min, centrifugation time is all 25 minutes;Centrifugation speed in step S8 It is 13000 revs/min to spend, and centrifugation time is 30 minutes;Centrifugal speed in step S9 is 13000 revs/min, and centrifugation time is 15 minutes.
CN201710183413.1A 2017-03-24 2017-03-24 A kind of method for extracting tan leather DNA Pending CN106754899A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710183413.1A CN106754899A (en) 2017-03-24 2017-03-24 A kind of method for extracting tan leather DNA

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710183413.1A CN106754899A (en) 2017-03-24 2017-03-24 A kind of method for extracting tan leather DNA

Publications (1)

Publication Number Publication Date
CN106754899A true CN106754899A (en) 2017-05-31

Family

ID=58967342

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710183413.1A Pending CN106754899A (en) 2017-03-24 2017-03-24 A kind of method for extracting tan leather DNA

Country Status (1)

Country Link
CN (1) CN106754899A (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011024439A (en) * 2009-07-22 2011-02-10 Tokyo Metropolitan Government Method for preparing sample nucleic acid
CN102199665A (en) * 2011-03-29 2011-09-28 镇江出入境检验检疫局检验检疫综合技术中心 LAMP (loop-mediated isothermal amplification) rapid detection kit and detection method for salmonella
CN102796826A (en) * 2012-09-07 2012-11-28 广州市质量监督检测研究院 Kit capable of quickly detecting real properties of leather and detecting method of kit
CN104004746A (en) * 2014-05-23 2014-08-27 青岛农业大学 Method for extracting maize single grain endosperm DNA capable of maintaining seed vigor
CN106119346A (en) * 2016-06-16 2016-11-16 广州市谱尼测试技术有限公司 A kind of method for identifying natural crocodile

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2011024439A (en) * 2009-07-22 2011-02-10 Tokyo Metropolitan Government Method for preparing sample nucleic acid
CN102199665A (en) * 2011-03-29 2011-09-28 镇江出入境检验检疫局检验检疫综合技术中心 LAMP (loop-mediated isothermal amplification) rapid detection kit and detection method for salmonella
CN102796826A (en) * 2012-09-07 2012-11-28 广州市质量监督检测研究院 Kit capable of quickly detecting real properties of leather and detecting method of kit
CN104004746A (en) * 2014-05-23 2014-08-27 青岛农业大学 Method for extracting maize single grain endosperm DNA capable of maintaining seed vigor
CN106119346A (en) * 2016-06-16 2016-11-16 广州市谱尼测试技术有限公司 A kind of method for identifying natural crocodile

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
《朴英实 等》: "《分子病理生物学实验技术指南》", 31 May 2015, 人民军医出版社 *
柯振华 等: "牛皮革PCR鉴别方法研究", 《皮革科学与工程》 *
童大跃 等: "《新编法医物证检验技术》", 30 November 2013, 中国医药科技出版社 *
赖心田 等: "皮革制品DNA属性鉴定技术的研究", 《中国皮革》 *
顾文佳 等: "基于PCR技术的水貂毛皮鉴别研究", 《纺织导报》 *

Similar Documents

Publication Publication Date Title
JP6366053B2 (en) Standard sample manufacturing method
CN109913447A (en) Free DNA extraction enrichment kit and free DNA extraction method
CN110628931B (en) Screening and application of eggplant SSR molecular marker core primer
CN107586887A (en) A kind of porcine circovirus 2 type and 3 type PCR differential diagnosis kits and its detection method
CN104370997B (en) Remove kit, the preparation method of method and its biological products of bacterial endotoxin in biological products
CN105861641A (en) Primer, kit and method for detecting CHO cell DNA residues
CN102605102A (en) One-tube magnetic bead method for extraction of virus nucleic acid for fluorescent quantitative detection
CN107201361A (en) A kind of extracting method of excretion body total serum IgE
CN108977437A (en) A kind of kit and its method extracting nucleic acid from serum plasma sample
CN101481742B (en) Detection kit for Mycoplasma hyopneumoniae and use thereof
CN103981285A (en) Detection method for infectious bovine viral diarrhea virus in aerosol
CN108949961A (en) For detecting kit and its screening of adenovirus pneumonia
CN109136389A (en) A kind of DNA bar code standard detection segment CO II, kit and its application method identifying American cockroach
CN103789197B (en) A kind of test kit and extracting method thereof extracting Microrna
CN106754899A (en) A kind of method for extracting tan leather DNA
CN109837331A (en) Detect the primer pair and method of Vero cellular DNA fragments size distribution
CN101875980A (en) Kit and method for detecting Macrobrachium rosenbergii Nodavirus
CN107475411A (en) A kind of Taqman real-time fluorescence PCR detection methods for detecting Borrelia burgdoyferi nucleic acid
CN102796826A (en) Kit capable of quickly detecting real properties of leather and detecting method of kit
CN111748638A (en) Specific primer, kit and method for identifying ips insects based on DNA bar code
CN110628951A (en) Fluorescence quantitative PCR (polymerase chain reaction) on-site rapid detection kit for African swine fever virus
CN111518878B (en) Method for evaluating animal model in screening of neoantigen vaccine or drug and application
JP4448673B2 (en) Bacteria analyzer and method
CN105586339B (en) It is a kind of identification Dysmicoccus neobrevipes Beardsley primer pair and its application
CN103667460A (en) A method for auxiliary detection of whether genetically modified plant components are contained in food

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20170531

RJ01 Rejection of invention patent application after publication