CN106701740A - Genomic DNA extraction kit and extraction method thereof - Google Patents
Genomic DNA extraction kit and extraction method thereof Download PDFInfo
- Publication number
- CN106701740A CN106701740A CN201611107846.0A CN201611107846A CN106701740A CN 106701740 A CN106701740 A CN 106701740A CN 201611107846 A CN201611107846 A CN 201611107846A CN 106701740 A CN106701740 A CN 106701740A
- Authority
- CN
- China
- Prior art keywords
- concentration
- gun head
- hcl
- liquid transfer
- tris
- 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
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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 invention provides a genomic DNA extraction kit and an extraction method thereof. The genomic DNA extraction kit comprises a digestion solution, a lysis solution, nanomagnetic beads, a washing liquid A, a washing liquid B and eluent. The extraction method provided by the invention has characteristics of simple extraction and purification steps, safe and convenient operation processes, high content of genomic DNA products, good integrity and high purity, can quickly digest and treat gastric mucosal samples within 1-2 hours, obtains complete high-purity genomic DNA through simple operation steps, and facilitates researchers to perform follow-up experiments.
Description
Technical field
The present invention relates to DNA extractive techniques field, and in particular to gastric mucosa tissue genome DNA extracting reagent kit and its carry
Take method.
Background technology
With the development of Protocols in Molecular Biology, the research in genomic level has turned into focus.In molecular genetics and
In molecular epidemiology, either whole-genome association (GWAS), candidate SNP locus Genotyping, or genome text
The foundation in storehouse, is required for extracting genomic DNA from Different categories of samples.Extract the DNA concentration, purity and the primary structure that obtain
Integrality can all have influence on follow-up research, thus efficiently, reliable genome DNA extracting method be that molecular genetics basis is ground
Study carefully in the urgent need to.The method of current extracting genome DNA purifying has a many kinds, such as alkaline lysis, boiling method, salting out method, from
Stem method and paramagnetic particle method.These methods respectively have advantage and disadvantage, but are all considered as following principle：Prevent and suppress drops of the DNase to DNA
Solution；The mechanical shearing destruction to DNA in solution is reduced as far as possible, keeps the complete of DNA molecular；By things such as protein, lipid, carbohydrates
Matter separates clean.
Alkaline lysis is denaturation and renaturation difference based on DNA and reaches and separate purpose, but the genomic DNA for obtaining contains
More impurity.Boiling method is that the DNA in sample is discharged by the effect of nucleic acid cleavage liquid by high-temperature boiling, centrifugation
By Impurity removal after precipitation, supernatant is that the template that can be used as some subsequent experimentals is used.Salting out method is using DNA and protein
The different solubility in electrolytic solution, the two is separated, and conventional method is extracted with 1M common salts, the DNP mucus that obtains with
Chloroform containing a small amount of octanol is swayed together, emulsifies it, then be centrifuged off protein.Now protein gel rests on water phase
And in the middle of chloroform phase, and DNA is located in upper strata aqueous phase, can be precipitated out DNA sodium salts with 2 times of ethanol of volume 95%.Centrifugal column
Method be genomic DNA under high salt conditions selective absorption in silicon substrate plasma membrane in centrifugal column, then by a series of quick rinsings+
The step of centrifugation, by cell metabolite, the Impurity removal such as albumen, finally using the elution buffer of less salt by pure genome
DNA is eluted from silicon substrate plasma membrane.
The nanometer magnetic bead that paramagnetic particle method is used is a kind of new functionalization solid carrier, the active base of its pan coating
Group, can be coupled with various bioactivators, with liquid mobility and solid magnetic material the features such as, outside
Can be with displacement and concentration, after external magnetic field is removed in the presence of magnetic field, slightly vibrating or aspirate can be dispersed in liquid again
In body, so that the separation change of solid liquid phase is very efficient and convenient, purity targeting very high can be obtained by simple wash-out
Material.For example, in the sample solution of abundant cracking, nanometer magnetic bead can with specific adsorption DNA, this kind of absorption specificity with
DNA structure and clip size are unrelated.Albumen and salt ion in removal solution is washed by cleaning solution, is finally only being left specifically
Eluent is added in the magnetic bead of adsorption of DNA, DNA is released in eluent.Paramagnetic particle method extracts genomic DNA and above-mentioned other sides
Method is compared, and does not use toxic reagent, simple to operate, is difficult pollution.
The content of the invention
In order to solve the defect of above-mentioned deficiency, the invention provides a kind of genome DNA extracting reagent kit and extracting method,
Fast with digestion process stomach lining sample speed, extraction purification step is few, easy to operate, and whole process is not related to toxic reagent,
Safe and convenient, gained Genome DNA content is high, and integrity degree is good, and purity is high, can be directly used for subsequent detection.
The invention provides a kind of genome DNA extracting reagent kit, including digestive juice, lysate, nanometer magnetic bead, cleaning solution
A, cleaning solution B and eluent.
Above-mentioned kit, wherein, the digestive juice includes Tris-HCL, EDETATE SODIUM, NaCL and SDS, the cracking
Liquid includes guanidinium isothiocyanate, trisodium citrate, Tween 20, and the nanometer magnetic bead is enclosed with the Fe of silica3O4, wash
Washing liquid A includes guanidinium isothiocyanate, Tris-HCL and absolute ethyl alcohol, and the cleaning solution B includes Tris-HCL, NaCL and anhydrous second
Alcohol, the eluent includes Tris-HCL, EDETATE SODIUM.
Above-mentioned kit, wherein, Tris-HCL, pH 8.0- of the digestive juice comprising pH 7.0-9.0 concentration 1-5mM
The EDETATE SODIUM of 9.0 concentration 3-7mM, concentration 50-200mM NaCL and 0.5%-3%SDS.
Above-mentioned kit, wherein, the lysate pH value be 4.0-7.0, the guanidinium isothiocyanate comprising concentration 3-5mM,
The trisodium citrate and 1%-5%Tween 20 of concentration 50-200mM.
Above-mentioned kit, wherein, the nanometer magnetic bead is enclosed with the Fe of silica3O4, a diameter of 200-
2000nm, concentration is 50-200mg/ml.
Above-mentioned kit, wherein, guanidinium isothiocyanate, pH 7.0-9.0 concentration of the cleaning solution A comprising concentration 1-4mM
The absolute ethyl alcohol of the Tris-HCL and 20%-50% of 10-50mM.
Above-mentioned kit, wherein, Tris-HCL, concentration of the cleaning solution B comprising pH 7.0-9.0 concentration 1-5mM
The absolute ethyl alcohol of 10-50mM NaCL and 50%-80%.
Above-mentioned kit, wherein, the eluent is the Tris-HCL and pH of 7.0-9.0 concentration 5-20mM comprising pH
The EDETATE SODIUM of 8.0-9.0 concentration 0.5-3mM.
Another side of the invention, present invention also offers a kind of extracting method of genome DNA extracting reagent kit, including with
Lower step：
Step S1：The fresh gastric mucosa tissue samples of 5-300mg are taken in 1.5ml centrifuge tubes, using aseptic liquid transfer gun head to
100-300ul digestive juices are added in centrifuge tube, concussion mixes 1min, 65 DEG C of water-bath 1-2h, shaken every 15min mix therebetween
1min, until gastric mucosa tissue sample digests completely；
Step S2：Using aseptic liquid transfer gun head to addition 300-500ul lysates and 50- in the centrifuge tube after water-bath
The nanometer magnetic bead 10-30ul of 200mg/ml, concussion mixes 1min, is stored at room temperature 10min；
Step S3：Centrifuge tube in step S2 is placed in 30s on magnetic frame, treats that nanometer magnetic bead is drawn to after tube wall completely,
Inhaled using aseptic liquid transfer gun head and abandon supernatant solution；
Step S4：Using aseptic liquid transfer gun head to 500-800ul cleaning solution A are added in step S3 centrifuge tubes, this is centrifuged
Pipe concussion mixes 1min, is placed in 30s on magnetic frame, treats that nanometer magnetic bead is drawn to after tube wall completely, is inhaled using aseptic liquid transfer gun head
Abandon supernatant solution；
Step S5：Using aseptic liquid transfer gun head to 500-800ul cleaning solution B are added in step S4 centrifuge tubes, this is centrifuged
Pipe concussion mixes 1min, is placed in 30s on magnetic frame, treats that nanometer magnetic bead is drawn to after tube wall completely, is inhaled using aseptic liquid transfer gun head
Supernatant solution is abandoned, room temperature is uncapped and dries 10-20min until no liquid is remained in pipe；
Step S6：Using aseptic liquid transfer gun head to addition 100-300ul eluents, 65 DEG C of water-bath 5- in step S5 centrifuge tubes
10min；
Step S7：The concussion of step S6 centrifuge tubes is mixed into 1min, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to completely
After tube wall, supernatant is drawn to new centrifuge tube using aseptic liquid transfer gun head, that is, obtain genomic DNA.
The present invention has advantages below：Extraction purification step is simple, safe operation process is convenient, genomic DNA product contains
Amount is high, and integrity degree is good, and purity is high.Stomach lining sample can be processed in 1-2 hours by rapid digestion, by shirtsleeve operation step
High-purity, complete genomic DNA are obtained, facilitates researcher to carry out follow-up experiment.
Brief description of the drawings
By the detailed description made to non-limiting example with reference to the following drawings of reading, the present invention and its feature, outward
Shape and advantage will become more apparent upon.Identical mark indicates identical part in whole accompanying drawings.Not deliberately proportionally
Draw accompanying drawing, it is preferred that emphasis is purport of the invention is shown.
Fig. 1 is that gastric mucosa tissue genome DNA extracting method of the invention is given birth to QIAamp DNA Mini Kit with Shanghai
The genomic DNA integrity degree comparison schematic diagram of work paramagnetic particle method genome DNA extraction kit (animal) extracting method.
Fig. 2 is that gastric mucosa tissue genome DNA extracting method of the invention is given birth to QIAamp DNA Mini Kit with Shanghai
β-actin fluorescent PCR Ct values compare in the genomic DNA of work paramagnetic particle method genome DNA extraction kit (animal) extracting method
Schematic diagram.
Specific embodiment
In the following description, a large amount of concrete details are given to provide more thorough understanding of the invention.So
And, it is obvious to the skilled person that the present invention can be able to without one or more of these details
Implement.In other examples, in order to avoid obscuring with the present invention, do not enter for some technical characteristics well known in the art
Row description.
In order to thoroughly understand the present invention, detailed step and detailed structure will be proposed in following description, so as to
Explaination technical scheme.Presently preferred embodiments of the present invention is described in detail as follows, but in addition to these detailed descriptions, this
Invention can also have other embodiment.
The invention provides a kind of genome DNA extracting reagent kit, including digestive juice, lysate, nanometer magnetic bead, cleaning solution
A, cleaning solution B and eluent.
In an of the invention preferred but unrestricted embodiment, digestive juice include Tris-HCL, EDETATE SODIUM, NaCL and
SDS, lysate includes guanidinium isothiocyanate, trisodium citrate, Tween 20, and the nanometer magnetic bead is enclosed with silica
Fe3O4, cleaning solution A include guanidinium isothiocyanate, Tris-HCL and absolute ethyl alcohol, the cleaning solution B include Tris-HCL, NaCL and
Absolute ethyl alcohol, eluent includes Tris-HCL, EDETATE SODIUM.
In a preferred but unrestricted embodiment of the invention, Tris- of the digestive juice comprising pH 7.0-9.0 concentration 1-5mM
The EDETATE SODIUM of HCL, pH 8.0-9.0 concentration 3-7mM, concentration 50-200mM NaCL and 0.5%-3%SDS.
In a preferred but unrestricted embodiment of the invention, lysate pH value is 4.0-7.0, comprising concentration 3-5mM's
Guanidinium isothiocyanate, the trisodium citrate of concentration 50-200mM and 1%-5%Tween 20.
In a preferred but unrestricted embodiment of the invention, nanometer magnetic bead is enclosed with the Fe of silica3O4, directly
Footpath is 200-2000nm, and concentration is 50-200mg/ml.
In a preferred but unrestricted embodiment of the invention, guanidinium isothiocyanate, pH of the cleaning solution A comprising concentration 1-4mM
The absolute ethyl alcohol of the Tris-HCL and 20%-50% of 7.0-9.0 concentration 10-50mM.
In a preferred but unrestricted embodiment of the invention, cleaning solution B includes pH 7.0-9.0 concentration 1-5mM's
The absolute ethyl alcohol of Tris-HCL, concentration 10-50mM NaCL and 50%-80%.
In a preferred but unrestricted embodiment of the invention, eluent is 7.0-9.0 concentration 5-20mM's comprising pH
The EDETATE SODIUM of Tris-HCL and pH 8.0-9.0 concentration 0.5-3mM.
A kind of extracting method of genome DNA extracting reagent kit of another side of the invention, comprises the following steps：
Step S1：The fresh gastric mucosa tissue samples of 5-300mg are taken in 1.5ml centrifuge tubes, using aseptic liquid transfer gun head to
100-300ul digestive juices are added in centrifuge tube, concussion mixes 1min, 65 DEG C of water-bath 1-2h, shaken every 15min mix therebetween
1min, until gastric mucosa tissue sample digests completely.
Step S2：Using aseptic liquid transfer gun head to addition 300-500ul lysates and 50- in the centrifuge tube after water-bath
The nanometer magnetic bead 10-30ul of 200mg/ml, concussion mixes 1min, is stored at room temperature 10min.
Step S3：Centrifuge tube in step S2 is placed in 30s on magnetic frame, treats that nanometer magnetic bead is drawn to after tube wall completely,
Inhaled using aseptic liquid transfer gun head and abandon supernatant solution.
Step S4：Using aseptic liquid transfer gun head to 500-800ul cleaning solution A are added in step S3 centrifuge tubes, this is centrifuged
Pipe concussion mixes 1min, is placed in 30s on magnetic frame, treats that nanometer magnetic bead is drawn to after tube wall completely, is inhaled using aseptic liquid transfer gun head
Abandon supernatant solution.
Step S5：Using aseptic liquid transfer gun head to 500-800ul cleaning solution B are added in step S4 centrifuge tubes, this is centrifuged
Pipe concussion mixes 1min, is placed in 30s on magnetic frame, treats that nanometer magnetic bead is drawn to after tube wall completely, is inhaled using aseptic liquid transfer gun head
Supernatant solution is abandoned, room temperature is uncapped and dries 10-20min until no liquid is remained in pipe.
Step S6：Using aseptic liquid transfer gun head to addition 100-300ul eluents, 65 DEG C of water-bath 5- in step S5 centrifuge tubes
10min。
Step S7：The concussion of step S6 centrifuge tubes is mixed into 1min, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to completely
After tube wall, supernatant is drawn to new centrifuge tube using aseptic liquid transfer gun head, that is, obtain genomic DNA.
Hereinafter enumerate specific embodiment of the invention.
Embodiment 1
1) the fresh gastric mucosa tissue samples of 5mg are taken in 1.5ml centrifuge tubes, is added in centrifuge tube using aseptic liquid transfer gun head
Enter 100ul digestive juices, concussion mixes 1min, 65 DEG C of water-bath 1h.1min is mixed every 15min concussions therebetween, until stomach lining group
Sample is knitted to digest completely.Wherein, the composition of addition is respectively in digestive juice：Tris-HCL, pH 8.1 of pH 7.5 and concentration 3mM
And EDETATE SODIUM, concentration the 80mM NaCL and 1.5%SDS of concentration 4.5mM, remaining composition is water.
2) using aseptic liquid transfer gun head to the nanometer magnetic bead 10ul that 300ul lysates and 50mg/ml are added in the centrifuge tube.
Nanometer magnetic bead can fully be mixed before plus nano magnetic bead.Concussion mixes 1min, is stored at room temperature 10min.Wherein, add in lysate
The composition for entering is respectively：The guanidinium isothiocyanate of concentration 3mM, the trisodium citrate of concentration 150mM and 2%Tween 20, remaining into
It is divided into water.The pH value of lysate is 6.0.
3) centrifuge tube is placed in 30s on magnetic frame, treats that nanometer magnetic bead is drawn to after tube wall completely, use aseptic liquid-transfering gun
Head is inhaled and abandons supernatant solution.
4) centrifuge tube is taken out from magnetic frame, using aseptic liquid transfer gun head to addition 500ul cleaning solution A in the centrifuge tube,
Centrifuge tube concussion is mixed into 1min, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to after tube wall completely, use aseptic shifting
Liquid pipette tips are inhaled and abandon supernatant solution.Wherein, the composition of addition is respectively in cleaning solution A：The guanidinium isothiocyanate of concentration 1mM, pH 7.5
And the absolute ethyl alcohol of the Tris-HCL of concentration 15mM and 50%, remaining composition is water.
5) centrifuge tube is taken out from magnetic frame, using aseptic liquid transfer gun head to addition 500ul cleaning solution B in the centrifuge tube,
Centrifuge tube concussion is mixed into 1min, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to after tube wall completely, use aseptic shifting
Liquid pipette tips are inhaled and abandon supernatant solution.Wherein, the composition of addition is respectively in cleaning solution B：The Tris-HCL of pH 7.5 and concentration 1mM,
The absolute ethyl alcohol of concentration 10mM NaCL and 80%, remaining composition is water.
6) centrifuge tube is taken out from magnetic frame, room temperature is uncapped and dries 10min until no liquid is remained in pipe.
7) using aseptic liquid transfer gun head to addition 100ul eluents, 65 DEG C of water-bath 10min in the centrifuge tube.Wherein, elute
The composition of addition is respectively in liquid：The EDETATE SODIUM of the Tris-HCL and pH 8.0 and concentration 2mM of pH 7.5 and concentration 5mM, its
Remaining composition is water.The pH value of eluent is 8.0.
8) centrifuge tube concussion is mixed into 1min, is placed in 30s on magnetic frame, treat that nanometer magnetic bead is drawn to after tube wall completely,
Supernatant is drawn to new centrifuge tube using aseptic liquid transfer gun head, that is, obtain genomic DNA.
Embodiment 2
1) the fresh gastric mucosa tissue samples of 300mg are taken in 1.5ml centrifuge tubes, using aseptic liquid transfer gun head in centrifuge tube
300ul digestive juices are added, concussion mixes 1min, 65 DEG C of water-bath 2h.1min is mixed every 15min concussions therebetween, until stomach lining
Tissue samples digest completely.Wherein, the composition of addition is respectively in digestive juice：Tris-HCL, pH of pH 7.5 and concentration 3mM
The EDETATE SODIUM of 8.1 and concentration 4.5mM, concentration 80mM NaCL and 1.5%SDS, remaining composition are water.
2) using aseptic liquid transfer gun head to the nanometer magnetic bead that 500ul lysates and 200mg/ml are added in the centrifuge tube
30ul.Nanometer magnetic bead can fully be mixed before plus nano magnetic bead.Concussion mixes 1min, is stored at room temperature 10min.Wherein, crack
The composition of addition is respectively in liquid：The guanidinium isothiocyanate of concentration 5mM, the trisodium citrate of concentration 150mM and 2%Tween 20,
Remaining composition is water.The pH value of lysate is 6.0.
3) centrifuge tube is placed in 30s on magnetic frame, treats that nanometer magnetic bead is drawn to after tube wall completely, use aseptic liquid-transfering gun
Head is inhaled and abandons supernatant solution.
4) centrifuge tube is taken out from magnetic frame, using aseptic liquid transfer gun head to addition 800ul cleaning solution A in the centrifuge tube,
Centrifuge tube concussion is mixed into 1min, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to after tube wall completely, use aseptic shifting
Liquid pipette tips are inhaled and abandon supernatant solution.Wherein, the composition of addition is respectively in cleaning solution A：The guanidinium isothiocyanate of concentration 1mM, pH 7.5
And the absolute ethyl alcohol of the Tris-HCL of concentration 15mM and 50%, remaining composition is water.
5) centrifuge tube is taken out from magnetic frame, using aseptic liquid transfer gun head to addition 800ul cleaning solution B in the centrifuge tube,
Centrifuge tube concussion is mixed into 1min, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to after tube wall completely, use aseptic shifting
Liquid pipette tips are inhaled and abandon supernatant solution.Wherein, the composition of addition is respectively in cleaning solution B：The Tris-HCL of pH 7.5 and concentration 1mM,
The absolute ethyl alcohol of concentration 10mM NaCL and 80%, remaining composition is water.
6) centrifuge tube is taken out from magnetic frame, room temperature is uncapped and dries 20min until no liquid is remained in pipe.
7) using aseptic liquid transfer gun head to addition 300ul eluents, 65 DEG C of water-bath 10min in the centrifuge tube.Wherein, elute
The composition of addition is respectively in liquid：The EDETATE SODIUM of the Tris-HCL and pH 8.0 and concentration 2mM of pH 7.5 and concentration 5mM, its
Remaining composition is water.The pH value of eluent is 8.0.
8) centrifuge tube concussion is mixed into 1min, is placed in 30s on magnetic frame, treat that nanometer magnetic bead is drawn to after tube wall completely,
Supernatant is drawn to new centrifuge tube using aseptic liquid transfer gun head, that is, obtain genomic DNA.
Embodiment 3
1) the fresh gastric mucosa tissue samples of 100mg are taken in 1.5ml centrifuge tubes, using aseptic liquid transfer gun head in centrifuge tube
200ul digestive juices are added, concussion mixes 1min, 65 DEG C of water-bath 2h.1min is mixed every 15min concussions therebetween, until stomach lining
Tissue samples digest completely.Wherein, the composition of addition is respectively in digestive juice：Tris-HCL, pH of pH 7.5 and concentration 3mM
The EDETATE SODIUM of 8.1 and concentration 4.5mM, concentration 80mM NaCL and 1.5%SDS, remaining composition are water.
2) using aseptic liquid transfer gun head to the nanometer magnetic bead that 300ul lysates and 100mg/ml are added in the centrifuge tube
10ul.Nanometer magnetic bead can fully be mixed before plus nano magnetic bead.Concussion mixes 1min, is stored at room temperature 10min.Wherein, crack
The composition of addition is respectively in liquid：The guanidinium isothiocyanate of concentration 5mM, the trisodium citrate of concentration 150mM and 2%Tween 20,
Remaining composition is water.The pH value of lysate is 6.0.
3) centrifuge tube is placed in 30s on magnetic frame, treats that nanometer magnetic bead is drawn to after tube wall completely, use aseptic liquid-transfering gun
Head is inhaled and abandons supernatant solution.
4) centrifuge tube is taken out from magnetic frame, using aseptic liquid transfer gun head to addition 500ul cleaning solution A in the centrifuge tube,
Centrifuge tube concussion is mixed into 1min, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to after tube wall completely, use aseptic shifting
Liquid pipette tips are inhaled and abandon supernatant solution.Wherein, the composition of addition is respectively in cleaning solution A：The guanidinium isothiocyanate of concentration 1mM, pH 7.5
And the absolute ethyl alcohol of the Tris-HCL of concentration 15mM and 50%, remaining composition is water.
5) centrifuge tube is taken out from magnetic frame, using aseptic liquid transfer gun head to addition 500ul cleaning solution B in the centrifuge tube,
Centrifuge tube concussion is mixed into 1min, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to after tube wall completely, use aseptic shifting
Liquid pipette tips are inhaled and abandon supernatant solution.Wherein, the composition of addition is respectively in cleaning solution B：The Tris-HCL of pH 7.5 and concentration 1mM,
The absolute ethyl alcohol of concentration 10mM NaCL and 80%, remaining composition is water.
6) centrifuge tube is taken out from magnetic frame, room temperature is uncapped and dries 20min until no liquid is remained in pipe.
7) using aseptic liquid transfer gun head to addition 100ul eluents, 65 DEG C of water-bath 10min in the centrifuge tube.Wherein, elute
The composition of addition is respectively in liquid：The EDETATE SODIUM of the Tris-HCL and pH 8.0 and concentration 2mM of pH 7.5 and concentration 5mM, its
Remaining composition is water.The pH value of eluent is 8.0.
8) centrifuge tube concussion is mixed into 1min, is placed in 30s on magnetic frame, treat that nanometer magnetic bead is drawn to after tube wall completely,
Supernatant is drawn to new centrifuge tube using aseptic liquid transfer gun head, that is, obtain genomic DNA.
In the present invention, contrast agents 1 extract the fresh gastric mucosa tissue samples of 20mg from QIAamp DNA Mini Kit
This, elution volume is 100ul.Contrast agents 2 are given birth to work paramagnetic particle method genome DNA extraction kit (animal) and are extracted from Shanghai
The fresh gastric mucosa tissue samples of 20mg, elution volume is 100ul.
By embodiment 3, contrast agents 1 and contrast agents 2, after the completion of extraction, Genomic DNA solution 5ul is taken respectively and is done
1.5% agarose gel electrophoresis, as a result as shown in Figure 1.
Shown in reference picture 1, the gastric mucosa tissue extracting genome DNA reagent based on nanometer magnetic bead set up using the present invention
Box and extracting method, by its experimental result and QIAamp DNA Mini Kit and Shanghai life work paramagnetic particle method genome DNA extraction examination
The Comparison of experiment results of agent box (animal), as a result shows, the present invention extract gastric mucosa tissue genomic DNA integrity degree with
QIAamp DNA Mini Kit and the genomic DNA integrity degree in Shanghai life work paramagnetic particle method genome DNA extraction kit (animal)
In same level.And with extraction purification step is simple, safe operation process is convenient.
By embodiment 3, contrast agents 1 and contrast agents 2, after the completion of extraction, Genomic DNA solution 2ul is taken respectively, use
The ultramicron bio-detectors of NanoDrop 2000 detection genomic DNA concentration and the purity of ThermoFisher, as a result such as table 1
It is shown.
Table 1
With reference to shown in table 1, purity and the QIAamp DNA Mini of the gastric mucosa tissue genomic DNA that the present invention is extracted
The genomic DNA purity in Kit and Shanghai life work paramagnetic particle method genome DNA extraction kit (animal) is in same level.For
The concentration of genomic DNA is also slightly better than contrast agents box.
By embodiment 3, contrast agents 1 and contrast agents 2, after the completion of extraction, Genomic DNA solution 5ul is taken respectively and makees target
The quantitative fluorescent PCR of β-action is designated as, as a result as shown in accompanying drawing 2 and table 2.
Table 2
Reference picture 2 and as shown in table 2, the present invention extract gastric mucosa tissue genomic DNA concentration apparently higher than
QIAamp DNA Mini Kit and the genomic DNA concentration in Shanghai life work paramagnetic particle method genome DNA extraction kit (animal).
Presently preferred embodiments of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, wherein the equipment and structure that do not describe in detail to the greatest extent are construed as giving reality with the common mode in this area
Apply；Any those of ordinary skill in the art, in the case where technical solution of the present invention ambit is not departed from, all using the disclosure above
Methods and techniques content make many possible variations and modification to technical solution of the present invention, or be revised as equivalent variations etc.
Effect embodiment, this has no effect on substance of the invention.Therefore, every content without departing from technical solution of the present invention, foundation
Technical spirit of the invention still falls within the present invention to any simple modification, equivalent variation and modification made for any of the above embodiments
In the range of technical scheme protection.
Claims (10)
1. a kind of genome DNA extracting reagent kit, it is characterised in that including digestive juice, lysate, nanometer magnetic bead, cleaning solution A,
Cleaning solution B and eluent.
2. a kind of genome DNA extracting reagent kit as claimed in claim 1, it is characterised in that the digestive juice includes Tris-
HCL, EDETATE SODIUM, NaCL and SDS, the lysate include guanidinium isothiocyanate, trisodium citrate, Tween 20, the nanometer
Magnetic bead is enclosed with the Fe of silica3O4, cleaning solution A includes guanidinium isothiocyanate, Tris-HCL and absolute ethyl alcohol, described to wash
Washing liquid B includes Tris-HCL, NaCL and absolute ethyl alcohol, and the eluent includes Tris-HCL, EDETATE SODIUM.
3. a kind of genome DNA extracting reagent kit as described in claim any one of 1-2, it is characterised in that the digestive juice
EDETATE SODIUM, the concentration 50-200mM of Tris-HCL, pH 8.0-9.0 concentration 3-7mM comprising pH 7.0-9.0 concentration 1-5mM
NaCL and 0.5%-3%SDS.
4. a kind of genome DNA extracting reagent kit as claimed in claim 3, it is characterised in that the lysate pH value is
4.0-7.0, the guanidinium isothiocyanate comprising concentration 3-5mM, the trisodium citrate of concentration 50-200mM and 1%-5%Tween 20.
5. a kind of genome DNA extracting reagent kit as claimed in claim 3, it is characterised in that the nanometer magnetic bead includes bag
It is wrapped with the Fe of silica3O4, a diameter of 200-2000nm, concentration is 50-200mg/ml.
6. a kind of genome DNA extracting reagent kit as claimed in claim 3, it is characterised in that the cleaning solution A includes concentration
The guanidinium isothiocyanate of 1-4mM, the absolute ethyl alcohol of the Tris-HCL and 20%-50% of pH 7.0-9.0 concentration 10-50mM.
7. a kind of genome DNA extracting reagent kit as claimed in claim 3, it is characterised in that the cleaning solution B includes pH
The Tris-HCL of 7.0-9.0 concentration 1-5mM, the absolute ethyl alcohol of concentration 10-50mM NaCL and 50%-80%.
8. a kind of genome DNA extracting reagent kit as claimed in claim 3, it is characterised in that the eluent is comprising pH
The EDETATE SODIUM of the Tris-HCL and pH 8.0-9.0 concentration 0.5-3mM of 7.0-9.0 concentration 5-20mM.
9. a kind of extracting method of genome DNA extracting reagent kit, it is characterised in that comprise the following steps：
Step S1：The fresh gastric mucosa tissue samples of 5-300mg are taken in 1.5ml centrifuge tubes, using aseptic liquid transfer gun head to centrifugation
100-300ul digestive juices are added in pipe, concussion mixes 1min, 65 DEG C of water-bath 1-2h；
Step S2：Using aseptic liquid transfer gun head to addition 300-500ul lysates and 50-200mg/ml in the centrifuge tube after water-bath
Nanometer magnetic bead 10-30ul, concussion mix 1min, be stored at room temperature 10min；
Step S3：Centrifuge tube in step S2 is placed in 30s on magnetic frame, treats that nanometer magnetic bead is drawn to after tube wall completely, used
Aseptic liquid transfer gun head is inhaled and abandons supernatant solution；
Step S4：Using aseptic liquid transfer gun head to 500-800ul cleaning solution A are added in step S3 centrifuge tubes, by centrifuge tube shake
Mixing 1min is swung, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to after tube wall completely, inhaled using aseptic liquid transfer gun head and abandoned
Clear solution；
Step S5：Using aseptic liquid transfer gun head to 500-800ul cleaning solution B are added in step S4 centrifuge tubes, by centrifuge tube shake
Mixing 1min is swung, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to after tube wall completely, inhaled using aseptic liquid transfer gun head and abandoned
Clear solution, room temperature is uncapped and dries 10-20min until no liquid is remained in pipe；
Step S6：Using aseptic liquid transfer gun head to addition 100-300ul eluents, 65 DEG C of water-bath 5- in step S5 centrifuge tubes
10min；
Step S7：The concussion of step S6 centrifuge tubes is mixed into 1min, 30s on magnetic frame is placed in, treats that nanometer magnetic bead is drawn to tube wall completely
Afterwards, supernatant is drawn to new centrifuge tube using aseptic liquid transfer gun head, that is, obtain genomic DNA.
10. a kind of extracting method of genome DNA extracting reagent kit as claimed in claim 9, it is characterised in that its feature exists
In also including in the step S1：1min is mixed every 15min concussions during 65 DEG C of water-bath 1-2h, until gastric mucosa tissue
Sample digests completely.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611107846.0A CN106701740A (en) | 2016-12-06 | 2016-12-06 | Genomic DNA extraction kit and extraction method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611107846.0A CN106701740A (en) | 2016-12-06 | 2016-12-06 | Genomic DNA extraction kit and extraction method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106701740A true CN106701740A (en) | 2017-05-24 |
Family
ID=58935994
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611107846.0A Pending CN106701740A (en) | 2016-12-06 | 2016-12-06 | Genomic DNA extraction kit and extraction method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106701740A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359665A (en) * | 2018-01-24 | 2018-08-03 | 安徽微分基因科技有限公司 | A kind of extracting method for high molecular weight genomic DNA in human blood based on kit |
| CN109694863A (en) * | 2019-02-28 | 2019-04-30 | 深圳市刚竹医疗科技有限公司 | Extracting method for the lysate of nucleic acid extraction, cleaning solution, nucleic acid extraction kit and nucleic acid |
| CN110452902A (en) * | 2018-05-08 | 2019-11-15 | 北京中科生仪科技有限公司 | A kind of Gram-negative bacteria complete genome DNA extracts kit |
| CN113046417A (en) * | 2021-03-25 | 2021-06-29 | 武汉吉诺百客医学科技有限公司 | Kit for extracting free DNA of blood plasma by paramagnetic particle method and use method thereof |
| CN113322253A (en) * | 2021-04-09 | 2021-08-31 | 长沙理工大学 | Kit for extracting genome DNA based on magnetic material and application method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613697A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | A kind of method of extracting purify DNA |
| CN101935645A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit for extracting DNA from histiocytes and method thereof |
| CN102220310A (en) * | 2009-08-05 | 2011-10-19 | 公安部物证鉴定中心 | Method for extracting and purifying spittle DNA |
| CN102888397A (en) * | 2012-09-25 | 2013-01-23 | 杭州硕航生物科技有限公司 | Kit using magnetic bead to extract whole blood genomic DNA and use of kit |
-
2016
- 2016-12-06 CN CN201611107846.0A patent/CN106701740A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101613697A (en) * | 2009-08-05 | 2009-12-30 | 公安部物证鉴定中心 | A kind of method of extracting purify DNA |
| CN102220310A (en) * | 2009-08-05 | 2011-10-19 | 公安部物证鉴定中心 | Method for extracting and purifying spittle DNA |
| CN101935645A (en) * | 2010-09-13 | 2011-01-05 | 原平皓(天津)生物技术有限公司 | Kit for extracting DNA from histiocytes and method thereof |
| CN102888397A (en) * | 2012-09-25 | 2013-01-23 | 杭州硕航生物科技有限公司 | Kit using magnetic bead to extract whole blood genomic DNA and use of kit |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108359665A (en) * | 2018-01-24 | 2018-08-03 | 安徽微分基因科技有限公司 | A kind of extracting method for high molecular weight genomic DNA in human blood based on kit |
| CN110452902A (en) * | 2018-05-08 | 2019-11-15 | 北京中科生仪科技有限公司 | A kind of Gram-negative bacteria complete genome DNA extracts kit |
| CN110452902B (en) * | 2018-05-08 | 2021-07-30 | 北京中科生仪科技有限公司 | Gram-negative bacterium whole genome DNA extraction kit |
| CN109694863A (en) * | 2019-02-28 | 2019-04-30 | 深圳市刚竹医疗科技有限公司 | Extracting method for the lysate of nucleic acid extraction, cleaning solution, nucleic acid extraction kit and nucleic acid |
| CN109694863B (en) * | 2019-02-28 | 2021-04-20 | 深圳市刚竹医疗科技有限公司 | Lysis solution for nucleic acid extraction, cleaning solution, nucleic acid extraction kit and nucleic acid extraction method |
| CN113046417A (en) * | 2021-03-25 | 2021-06-29 | 武汉吉诺百客医学科技有限公司 | Kit for extracting free DNA of blood plasma by paramagnetic particle method and use method thereof |
| CN113322253A (en) * | 2021-04-09 | 2021-08-31 | 长沙理工大学 | Kit for extracting genome DNA based on magnetic material and application method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106701740A (en) | Genomic DNA extraction kit and extraction method thereof | |
| CN103820431B (en) | Nucleic acid extraction purification process and kit based on nanometer magnetic bead | |
| CN106867992A (en) | A kind of whole blood DNA extracts kit and extracting method | |
| Emaus et al. | Nucleic acid extraction: Fundamentals of sample preparation methodologies, current advancements, and future endeavors | |
| CN107043766A (en) | A kind of nucleic acid magnetic bead extracts reagent of instant | |
| CN108624586B (en) | Nucleic acid extraction kit and application method thereof | |
| WO2017193832A1 (en) | Mitochondrial genomic library based on high-throughput sequencing and method for constructing the library | |
| JP2011505139A (en) | Method for isolating genomic DNA, RNA and protein from a single sample | |
| EP3904532A1 (en) | Nucleic acid extraction composition, reagent and kit containing the same and use thereof | |
| CN105695450A (en) | Magnetic-bead-process-based kit for extracting free DNAs (deoxyribonucleic acids) and application method thereof | |
| Wang et al. | Simultaneous detection of bovine rotavirus, bovine parvovirus, and bovine viral diarrhea virus using a gold nanoparticle-assisted PCR assay with a dual-priming oligonucleotide system | |
| CN113151397A (en) | Nucleic acid extraction kit for extracting virus sample based on paramagnetic particle method | |
| CN107794260A (en) | A kind of method that free nucleic acid is extracted in the acellular body fluid from large volume | |
| CN102911932A (en) | Assay kit and method for simultaneously extracting and purifying ribose nucleic acid (RNA) and deoxyribose nucleic acid (DNA) | |
| CN109762874B (en) | Nucleic acid sedimentation aid, pregnant woman plasma free DNA extraction kit and method | |
| CN106867996A (en) | A kind of cfDNA libraries of the method, kit and structure that cfDNA is extracted from hydrothorax | |
| CN105950610A (en) | Method for rapidly extracting fecal bacterial genome DNA and virus RNA through paramagnetic particle method | |
| CN112391381A (en) | Urine free DNA extraction kit based on nano magnetic beads and extraction method | |
| CN112899266A (en) | Cracking binding solution for nucleic acid extraction, kit and application thereof | |
| CN112195175A (en) | Nucleic acid extraction method based on graphene oxide | |
| CN113462683A (en) | Alcohol-free cleaning solution suitable for extracting nucleic acid from multiple samples and nucleic acid extraction kit | |
| CN103184214B (en) | A kind of hbv nucleic acid rapid extraction reagent | |
| CN115637267A (en) | Nucleic acid extraction reagent, kit and extraction method thereof | |
| WO2021244469A1 (en) | Nucleic acid extraction method and application | |
| CN110628762A (en) | Nucleic acid extraction method based on nano magnetic beads and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170524 |
|
| WD01 | Invention patent application deemed withdrawn after publication |