CN109371013A - It is a kind of from insect material extract DNA extractant and its application - Google Patents
It is a kind of from insect material extract DNA extractant and its application Download PDFInfo
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Abstract
The invention discloses a kind of extractant extracted for DNA and its applications.This is made of for DNA extractant extracted Tris-base, NaCl, PVP and Tween-20, can the micro insect material DNA of rapidly extracting, and only need to configure the extractant in sample extraction process, do not need to configure other liquid.Reagent used by extractant of the present invention is that chemical reagent is often used in laboratory, low in cost, save the cost；And extractant stable components, be not required to matching while using, can Reusability, be stored at room temperature at least three moon.The micro insect sample DNA process of present invention extraction simultaneously is easy to operate, quick, can complete to extract in 30s.The DNA mass that the present invention extracts is stablized, and directly can be used for subsequent experimental detection.The method great advantage is using only a kind of extracting solution, and extraction process can be completed in 30s, and DNA extracts quality and stablizes, and may be directly applied to subsequent experimental detection.
Description
Technical field
The invention patent relates to field of biotechnology, in particular to it is a kind of for insect minim DNA extract extractant and
It is applied.
Background technique
With modern molecular biology, the development of informatics, the various technique of gene detection based on DNA are quickly grown, such as
Specificity allele PCR amplification (allele specific amplification, ASA), restriction fragment length polymorphism
It analyzing (Restrietion fragment length polymorphism, RFLP), PCR is single-stranded/double-stranded conformational polymorphism
(single/double strand conformation polymorphism, SSCP), random amplified polymorphic DNA technique
(randomly amplified polymorphic DNA, RAPD), loop-mediated isothermal amplification technique (Loop-mediated
Isothermal amplification, LAMP) etc., it is widely used in caste identification identification, Detection of insecticide resistance monitoring, elder brother
The fields such as worm biological characteristic research protect the biological diversity of insect, harmful insect science to administer, beneficial insect conjunction for China
The reason theoretical foundation important using offer.And insect sample DNA extracting method is to realize a weight of these detection techniques application
Want condition.From development trend, more simple and quick, specific higher, more economical practical DNA extraction method is that necessity becomes
Gesture has broad application prospects.Traditional DNA method has very much, such as phenol chloroform method of traditional classical etc., these methods
The DNA purity of acquisition is very high, can satisfy the requirement of various tests, but cumbersome, with duration, and agents useful for same have it is certain
Toxicity.Such as salt extracting method, yield is relatively low, and purity is poor.Such as pellosil adsorption column method, paramagnetic particle method need repeatedly
Elution, operation are relatively still complicated.Domestic and international biotech firm also develops the nucleic acid DNA extracts kit of a variety of commercializations,
There is company to develop fast trace DNA method, but still exist generally operate it is more complex, need the time is long, sample need it is more,
The problems such as extracting unstable result.Therefore a kind of quick, easy, economic and stable DNA method is needed, to push above-mentioned detection
The application that technology is more simplified.
Summary of the invention
In view of this, the purpose of the present invention is to provide it is a kind of for insect trace sample DNA extract extractant and its
Application method and application.
To achieve the above object, the technical solution of the present invention is as follows:
A kind of extractant extracted for DNA, is the buffer being grouped as by following groups:
The pH of the buffer is 8.0, and solvent is distilled water.
The polyvinylpyrrolidone (PVP) is preferably polyvinylpyrrolidone K 40 (PVP K-40).
In of the invention one preferred technical solution, the concentration of the Tris-base is 25mM.
In another preferred technical solution of the invention, the concentration of the NaCl is 150mM.
In the preferred technical solution of another of the invention, the concentration of the polyvinylpyrrolidone is 0.02g/ml.
Application of the above-mentioned extractant in the DNA for micro insect sample is extracted also belongs to protection model of the invention
It encloses.
In the application, the micro insect sample refers to that insect specimen weight is 0.1-10mg.
It is to extract insect sample using above-mentioned extractant the present invention also provides a kind of method for extracting insect sample DNA
DNA。
In the method, the insect sample size is 0.1-10mg.
In an embodiment of the invention, the method includes the following steps:
0.1-10mg insect sample is added in the above-mentioned extractant of 10-100 μ L, grinds 5-7 seconds, vortex 2-3 seconds, is centrifuged
2-3 seconds, obtained supernatant was insect sample DNA solution.
In another embodiment of the invention, the method includes the following steps:
0.1-10mg insect sample is added in the above-mentioned extractant of 10-100 μ L, 5-7s is ground, 3-15s is stood, obtains
Insect sample DNA.
In the above method, the usage amount amount of the extractant is preferably 10-50 μ L insect sample, more preferable 20-50 μ L.
Extractant in the present invention is added PVP and Tween-20, is only configured a kind of extraction based on salt extracting method
Liquid simplifies extraction process, while the influence of removal sugar, fat, protein to greatest extent, and uses the extracting solution in practical field
When, it is only necessary to after standing sample after grinding, directly absorption supernatant is used for subsequent detection as DNA sample, improves in the present invention
The practical ranges of extracting solution and application method.The present invention quick from micro insect sample, easy can extract genome
DNA is to meet the molecular detection technology application request based on DNA.
The present invention is made of for the DNA extractant extracted Tris-base, NaCl, PVP and Tween-20, can quickly be mentioned
Micro insect material DNA is taken, and only needs to configure the extractant in sample extraction process, does not need to configure other liquid.This
Reagent used by invention extractant is that chemical reagent is often used in laboratory, low in cost, save the cost；And extractant ingredient is steady
It is fixed, be not required to matching while using, can Reusability, be stored at room temperature at least three moon.The present invention extracts micro insect sample DNA mistake simultaneously
Journey is easy to operate, quick, can complete to extract in 30s.The DNA mass that the present invention extracts is stablized, and directly can be used for subsequent
Experiment detection.The method great advantage is using only a kind of extracting solution, and extraction process can be completed in 30s, and DNA extracts quality
Stablize, may be directly applied to subsequent experimental detection.
It is demonstrated experimentally that the micro insect sample based on above technical scheme preparation, for originating insect specimen weight range
For 0.1-10mg, DNA concentration >=260/280 range of 150ng/ μ L, OD is extracted between 1.70-2.00.Compared with conventional method and
Existing kit method in the market, extractant of the invention and method are time saving and energy saving, fast, economical, avoid organic solvent dirty
Dye, and DNA mass is stablized, and subsequent detection demand is met.To realize that quick, the practical application of various detection methods provide important base
Plinth.
Detailed description of the invention
Fig. 1 is Tris alkali (X1), and NaCl (X2), PVP (X3) concentration is in three levels (1,2,3) to DNA concentration (A) and matter
Measure the influence of (B)
Fig. 2 is that the purchase using phenol chloroform method (A), the salting-out method (B) of comparative example 2, comparative example 3 of comparative example 1 tries
Agent box (C), the extractant of the invention of embodiment 1 and method (D) extract 24 black peach aphids (list, average weight 0.248m respectively
G) agarose gel electrophoresis figure of genomic DNA, M are DNA Marker.
Fig. 3, single black peach aphid DNA concentration (A) and purity (B) box traction substation are extracted using four kinds of methods.
Fig. 4, the agarose that PCR amplification purpose band is carried out using the black peach aphid single head DNA extracted using four kinds of methods as template
Gel electrophoresis figure, the DNA that (A) is extracted using phenol chloroform method shown in comparative example 1 is template；(B) with salt extraction side shown in comparative example 2
The DNA that method is extracted is template；(C) using the DNA that purchase kit extracts shown in comparative example 3 as template；(D) to utilize embodiment
The DNA that extractant of the invention shown in 1 and method are extracted is template, and M is DNA Marker.
Fig. 5, black peach aphid is extracted using two kinds of extracting methods of extractant optimal case (embodiment 1 and embodiment 7) in the present invention
The agarose gel electrophoresis figure of genomic DNA, (A) embodiment 1；(B) embodiment 7；M is DNA Marker.
Fig. 6, the single head black peach aphid DNA extracted in two ways are the Ago-Gel that template carries out PCR amplification purpose band
Electrophoretogram, the DNA that (A) is extracted using embodiment 1 is template；(B) DNA extracted using embodiment 7 is template；M is DNA Marker.
Specific embodiment
Gather specific embodiment below to further describe the invention patent, but is not that the contents of the present invention are only limitted to institute
It gives an actual example.So those skilled in the art carry out nonessential improvement and tune to embodiment according to foregoing invention content
It is whole, still fall within protection scope of the present invention.
One, material used in present embodiment is as follows:
1. experimental material: black peach aphid (only single, average weight 0.248m g), cotten aphid (single only, average weight 0.152mg),
Wheat aphid (single, average weight 0.294mg).
2. experiment reagent:
Trihydroxy aminomethane (Tris), ethylenediamine tetra-acetic acid (EDTA), lauryl sodium sulfate (SDS), polyethylene pyrrole
Pyrrolidone (PVP), sodium chloride, phenol, chloroform, hydrochloric acid, ethidium bromide EtBr, Tween-20 are AMRESCO Products.
RNase A, Proteinase K, DNA Marker DL1,000, DNAMarker DL10,000 are TAKARA Products.Agarose
(BIOWEST AGROSE) is GENE TECH Products.
3. laboratory apparatus
X-Gel gel imaging system instrument, microplate reader (TECAN infinite M200pro), Nanodrop (Thermo
Scientific, USA), DYY-6C type electrophoresis apparatus and electrophoresis tank (Liuyi Instruments Plant, Beijing), high speed freezing centrifuge
Centrifugue 5417R(Eppendorf)。
4. preparation of reagents
1) TNE buffer
10mM Tris.HCl(pH 7.5)
60mM NaCl
10mM EDTA(pH 8.0),
2) TNES buffer
50mM Tris.HCl(pH7.5)
400mM NaCl
20mM EDTA
0.5%SDS
3) TE buffer
10mM Tris.HCl
1mM EDTA(pH 8.0)
Two, the quick extractant that DNA is extracted from micro insect material of the invention obtains and optimizes:
The present inventor has found that the existing method for extracting DNA is excessively complicated in long-term research work, uncomfortable
DNA in requisition for the small insects quickly detected is extracted, it is therefore desirable to a kind of quick, easy, economic and stable DNA method,
With the application for pushing above-mentioned detection technique more to simplify.
Based on this, inventor gradually carries out trial screening to the reagent for being suitable for the extraction of micro insect sample DNA, finds Tris
Alkali, NaCl, PVP are fine to micro insect sample DNA sample effect is extracted, also, extraction process is very simple, rapidly.Wherein,
PVP is optimal using polyvinylpyrrolidone K 40 (PVP K-40) effect.
Inventor optimizes each component concentration by orthogonal experiment, and it is described that the specific method is as follows:
In order to evaluate the Tris alkali in the buffer formulation, the influence of NaCl, PVP to black peach aphid DNA concentration and quality is extracted
(Tween-20 is nonionic surfactant in the buffer, it acts as detergent, according to Literature Consult its to extracting DNA
The influence of quality and concentration is smaller), each 3 horizontal orthogonal tests of this 3 factors are carried out, according to L9(33) orthogonal arrage sets altogether
It is as follows to count 9 processing of DNA Extraction buffer:
1 L of table9(33) orthogonal table
Black peach aphid DNA extraction is carried out with above-mentioned 9 kinds of Extraction buffers respectively, it is described that the specific method is as follows:
1. single black peach aphid (single, average weight 0.248m g) is put into 1.5ml centrifuge tube, 30 μ L extracting solutions are added, grind
Grind 5s；
2. spiral 3s, rapid centrifugation 2s remove impurity.
3. taking 20 μ L supernatants as DNA sample, 4 degree of refrigerator are placed in case detection.
To its concentration and quality determination result and variance analysis.From the point of view of determination data, the DNA matter of 9 kinds of buffers extraction
Amount and concentration are all satisfied in subsequent experimental requirement.Wherein Tris alkali concentration is DNA concentration and the principal element that quality influences, PVP
Concentration is affected to DNA mass, and NaCl concentration is smaller (as shown in Figure 1) on DNA concentration and quality influence, and 9 kinds slow
The DNA concentration and quality the results of analysis of variance that fliud flushing is extracted are as shown in table 2, analyze in conjunction with table 1, and wherein Tris alkali concentration is 25mM
When, DNA concentration and quality are preferable, therefore Tris alkali concentration is set as 25mM in optimal buffer.And it is balanced consider NaCl and
Influence and economic cost of the PVP to DNA mass and concentration, NaCl concentration set 150mM, and the concentration of PVP is 0.02g/ml.
The DNA concentration and quality the results of analysis of variance that 29 kinds of buffers of table extract
Three, specific embodiment
Embodiment 1, extractant of the invention and its application
The extractant of the present embodiment is the buffer being grouped as by following groups:
The pH of the buffer is 8.0, and solvent is distilled water.
0.3028g Tris-base is weighed, 0.8775g NaCl is dissolved in 80mL distilled water, extremely with concentrated hydrochloric acid tune pH
8.0, polyvinylpyrrolidone K 40 (PVP K-40) 2g is added, 1mL Tween-20 is eventually adding distilled water and is settled to
100mL。
DNA extraction is carried out to black peach aphid sample using extractant of the invention, it is described that the specific method is as follows:
1. single black peach aphid (single, average weight 0.248m g) is put into 1.5ml centrifuge tube, 30 μ L extracting solutions are added, grind
Grind 5s；
2. spiral 3s, rapid centrifugation 2s remove impurity.
3. taking 20 μ L supernatants as DNA sample, 4 degree of refrigerator are placed in case detection.
Embodiment 2, extractant of the invention and its application (buffer 1 in orthogonal test designs table 1)
The extractant of the present embodiment is by the following groups of buffers being grouped as (in table 1 No. 1):
The pH of the buffer is 8.0, and solvent is distilled water.
The preparation method of extractant are as follows:
0.3028g Tris-base is weighed, 0.2925g NaCl is dissolved in 80mL distilled water, extremely with concentrated hydrochloric acid tune pH
8.0, polyvinylpyrrolidone K 40 (PVP K-40) 1g is added, 1mL Tween-20 is eventually adding distilled water and is settled to
100mL。
DNA extraction is carried out to black peach aphid sample using extractant of the invention, it is described that the specific method is as follows:
1. single black peach aphid (single, average weight 0.248m g) is put into 1.5ml centrifuge tube, 30 μ L extracting solutions are added, grind
Grind 5s；
2. spiral 3s, rapid centrifugation 2s.
3. taking 20 μ L supernatants as DNA sample, 4 degree of refrigerator are placed in case detection.
Embodiment 3, extractant of the invention and its application (buffer 5 in orthogonal test designs table 1)
The extractant of the present embodiment is by the following groups of buffers being grouped as (in table 1 No. 5):
The pH of the buffer is 8.0, and solvent is distilled water.
The preparation method of extractant are as follows:
0.6057g Tris-base is weighed, 0.5850g NaCl is dissolved in 80mL distilled water, extremely with concentrated hydrochloric acid tune pH
8.0, polyvinylpyrrolidone K 40 (PVP K-40) 4g is added, 1mL Tween-20 is eventually adding distilled water and is settled to
100mL。
DNA extraction is carried out to black peach aphid sample using extractant of the invention, it is described that the specific method is as follows:
1. single black peach aphid (single, average weight 0.248m g) is put into 1.5ml centrifuge tube, 30 μ L extracting solutions are added, grind
Grind 5s；
2. spiral 3s, rapid centrifugation 2s.
3. taking 20 μ L supernatants as DNA sample, 4 degree of refrigerator are placed in case detection.
Embodiment 4, extractant of the invention and its application (buffer 9 in orthogonal test designs table 1)
The extractant of the present embodiment is the buffer being grouped as by following groups:
The pH of the buffer is 8.0, and solvent is distilled water.
The preparation method of extractant are as follows:
0.9085g Tris-base is weighed, 1.1700g NaCl is dissolved in 80mL distilled water, extremely with concentrated hydrochloric acid tune pH
8.0, polyvinylpyrrolidone K 40 (PVP K-40) 1g is added, 1mL Tween-20 is eventually adding distilled water and is settled to
100mL。
DNA extraction is carried out to black peach aphid sample using extractant of the invention, it is described that the specific method is as follows:
1. single black peach aphid (single, average weight 0.248m g) is put into 1.5ml centrifuge tube, 30 μ L extracting solutions are added, grind
Grind 5s；
2. spiral 3s, rapid centrifugation 2s.
3. taking 20 μ L supernatants as DNA sample, 4 degree of refrigerator are placed in case detection.
Embodiment 5, extractant of the invention and its application
The extractant of the present embodiment is optimal case, is the buffer being grouped as by following groups:
The pH of the buffer is 8.0, and solvent is distilled water.
0.3028g Tris-base is weighed, 0.8775g NaCl is dissolved in 80mL distilled water, extremely with concentrated hydrochloric acid tune pH
8.0, polyvinylpyrrolidone K 40 (PVP K-40) 2g is added, 1mL Tween-20 is eventually adding distilled water and is settled to
100mL。
DNA extraction is carried out to cotten aphid sample using extractant of the invention, it is described that the specific method is as follows:
1. single cotten aphid (single, average weight 0.152mg) is put into 1.5ml centrifuge tube, 30 μ L extracting solutions are added, grind
Grind 6s；
2. spiral 3s, rapid centrifugation 2s.
3. taking 20 μ L supernatants as DNA sample, 4 degree of refrigerator are placed in case detection.
Embodiment 6, extractant of the invention and its application
The extractant of the present embodiment is optimal case, is the buffer being grouped as by following groups:
The pH of the buffer is 8.0, and solvent is distilled water.
0.3028g Tris-base is weighed, 0.8775g NaCl is dissolved in 80mL distilled water, extremely with concentrated hydrochloric acid tune pH
8.0, polyvinylpyrrolidone K 40 (PVP K-40) 2g is added, 1mL Tween-20 is eventually adding distilled water and is settled to
100mL。
DNA extraction is carried out to black peach aphid sample using extractant of the invention, it is described that the specific method is as follows:
1. single wheat aphid (single, average weight 0.294mg) is put into 1.5ml centrifuge tube, 30 μ L extracting solutions are added, grind
Grind 6s；
2. spiral 3s, rapid centrifugation 2s.
3. taking 20 μ L supernatants as DNA sample, 4 degree of refrigerator are placed in case detection.
Embodiment 7, extractant of the invention and its application
The extractant of the present embodiment is the buffer being grouped as by following groups:
The pH of the buffer is 8.0, and solvent is distilled water.
0.3028g Tris-base is weighed, 0.8775g NaCl is dissolved in 80mL distilled water, extremely with concentrated hydrochloric acid tune pH
8.0, polyvinylpyrrolidone K 40 (PVP K-40) 2g is added, 1mL Tween-20 is eventually adding distilled water and is settled to
100mL。
DNA extraction is carried out to black peach aphid sample using extractant of the invention, it is described that the specific method is as follows:
Single black peach aphid (single, average weight 0.248m g) is put into 1.5ml centrifuge tube, 30 μ L extracting solutions, grinding is added
5s；5s is stood, insect sample DNA is obtained.
Comparative example 1, phenol chloroform method extract insect DNA
1, single black peach aphid (single average weight 0.248m g) is put into 1.5ml centrifuge tube, 0.25mlTNE buffering is added
Liquid grinds 30s.
2, the SDS solution that 25 μ l mass percentage concentrations are 20% is added, spiral concussion mixes.
3,10 μ l Proteinase Ks (20mg/ml) are added, spiral concussion mixes, and is placed in 50 DEG C of water-baths and stays overnight.
4, the phenol of 250 μ l: chloroform (volume ratio 1:1) mixed liquor is added, room temperature shaker shakes 30min, room temperature
13000rpm is centrifuged 1min, and careful supernatant of drawing is transferred in new 1.5ml centrifuge tube.
5, it is primary to repeat step 4.
6,250 μ l chloroforms are added, room temperature shaker shakes 30min, and room temperature 13000rpm is centrifuged 1min, careful to draw supernatant extremely
In new 1.5ml centrifuge tube.
7,1.2 μ l RNase A (20mg/ml), 37 DEG C of incubation 60min. are added
8,5 μ l Proteinase Ks (20mg/ml) are added, 50 DEG C of DEG C of incubation 60min are mixed primary every the soft spiral of 20min.
9,10 μ l 5M NaCl solutions are added, mix.
10, after the 500 cold dehydrated alcohols of μ l of addition (- 20 DEG C of storages) are mixed by inversion, it is placed in -80 DEG C of DEG C of ultra low temperature freezers
20min。
10, sample is taken out, 4 DEG C DEG C, 13000rpm is centrifuged 10min.
11, supernatant is carefully removed, precipitating is left, 70% ethanol solution of concentration expressed in percentage by volume of 500 μ l pre-cooling is added, is resuspended
Precipitating.
12,4 DEG C DEG C, 13000rpm is centrifuged 10min, carefully removes supernatant, is deposited in air drying 5-10min.
13,30 μ l TE buffer solutions precipitating is added, 4 degree of refrigerator are placed in case detection.
Comparative example 2, salt extracting method extract insect DNA:
1. single black peach aphid (single average weight 0.248m g) is put into 1.5ml centrifuge tube, and pour into a small amount of liquid nitrogen.
2. 10 μ l Proteinase Ks (20mg/ml) are added, rapidly after grinding, 300 μ l TNES buffers are added.
3. as incubation 4h in 55 DEG C of water-baths or overnight after mixing.
4. 85 μ l 5M NaCl solutions are added, mix rapidly until there is albumen precipitation precipitation.
5. sample room temperature 13000rpm is centrifuged 15min.
6. carefully transfer supernatant is added the 400 cold dehydrated alcohols of μ l (- 20 DEG C of storages) and is mixed by inversion into new centrifuge tube
Afterwards, it is placed in 20min in -80 DEG C of DEG C of ultra low temperature freezers.
10, sample is taken out, 4 DEG C, 13000rpm is centrifuged 10min.
11, supernatant is carefully removed, precipitating is left, the concentration expressed in percentage by volume that 500 μ l pre-cooling is added is 70% ethanol solution, weight
Outstanding precipitating.
12,4 DEG C, 13000rpm is centrifuged 5min, carefully removes supernatant, is deposited in air drying 5-10min.
13,30 μ l TE buffer solutions precipitating is added, 4 degree of refrigerator are placed in case detection.
Comparative example 3, kit method extract insect DNA
(Tiangeng is raw using Fast DNA extraction detection kit TIANcombi DNA Lyse&Det PCRKit for this comparative example
Object Technology Co., Ltd., Beijing, article No. KG203) extract insect DNA.
1, single black peach aphid (single average weight 0.248m g) is put into the centrifuge tube of 1.5ml, the 30 above-mentioned examinations of μ l are added
Buffer solution B 1 in agent box, it is ensured that sample can be completely covered in buffer.
2, sample is smashed to pieces with grinding pestle.
3, after sample is smashed to pieces, the buffer solution B 2 in 30 μ l mentioned reagent boxes is added, concussion mixes.
4,12,000rpm (~13,400 × g) are centrifuged 2min.
5, after centrifugation, careful 30 μ l supernatants of drawing are used as template spare in another clean 1.5ml centrifuge tube.
Four, embodiment 1-7 and comparative example result statistics and analysis:
As shown in table 3, the optimization formula (embodiment 1) and other formula (embodiments of extractant in the present invention is respectively adopted
2-4), comparative example (1-3) extracts the genomic DNA of 100 black peach aphids respectively.Simultaneously using the optimal side of extractant in the present invention
Case has carried out DNA extraction to 50 cotten aphids (embodiment 5) and 50 wheat aphids (embodiment 6).It has also carried out mentioning in the present invention simultaneously
The optimal case of agent is taken to carry out the comparison of two kinds of embodiments.And integrality (the figure of DNA is had detected using agarose electrophoresis
2) DNA concentration and purity (table 1), are determined using microplate reader, having carried out case with Excel software to determination data must scheme to draw,
To analyze the DNA concentration and purity profile situation (Fig. 3) of 100 black peach aphids of every kind of method extraction, and had detected using PCR method
Extract applicability (Fig. 4) of the DNA in subsequent detection.1 detection data of complex chart 2 and table, traditional phenol chloroform method are extracted
The integrality and concentration of DNA is higher, but protein contamination is heavier, and complicated for operation, and time-consuming.And kit method extracts DNA's
Concentration is lower.Salting-out method extracts DNA concentration and purity belongs to the medium level of four kinds of methods, but complicated for operation, time-consuming.And this
The extracting solution and application method of invention can achieve the DNA extracting concentration and purity with salting-out method phase same level, from concentration numbers
According to, due to simplifying extraction step, the concentration of DNA and the stability of purity are significantly better than other three kinds of sides from the point of view of purity data
Method (Fig. 3).It, as can be seen from Figure 4 can be direct using the DNA that extracting solution of the invention and method obtain and in subsequent detection
It is detected for PCR, the DNA mass of extraction is highly stable.Meanwhile the present invention in extractant two kinds of embodiments to black peach aphid gene
Group DNA extracts quality and concentration has no significant effect, and in the application of subsequent detection technique also without different (Fig. 5 and Fig. 6)
Table 3, the DNA concentration and quality testing table that various methods are extracted
Described above to be merely exemplary for the purpose of the present invention, and not restrictive, those of ordinary skill in the art understand,
In the case where not departing from spirit and scope as defined in the appended claims, many modifications, variation or equivalent can be made, but all
It will fall within the scope of protection of the present invention.
Claims (10)
1. a kind of extractant extracted for DNA, which is characterized in that the extractant is the buffer being grouped as by following groups:
1)Tris-base 25-75mM；
2)NaCl 50-200mM；
3) polyvinylpyrrolidone 0.01-0.04g/ml；
4) Tween-20 concentration expressed in percentage by volume is 1%；
The pH of the buffer is 8.0, and solvent is water.
2. extractant according to claim 1, it is characterised in that: the concentration of the Tris-base is 25mM.
3. extractant according to claim 1, it is characterised in that: the concentration of the NaCl is 150mM.
4. extractant according to claim 1, it is characterised in that: the concentration of the polyvinylpyrrolidone is 0.02g/
ml。
5. application of the extractant described in any one of claim 1-4 in the DNA for micro insect sample is extracted.
6. application according to claim 5, mentions and being characterized in that: the micro insect sample refers to that insect specimen weight is
0.1-10mg。
7. a kind of method for extracting insect sample DNA, it is characterised in that: using being mentioned described in any one of claim 1-5
Agent is taken to extract insect sample DNA.
8. according to the method described in claim 7, it is characterized by: the insect sample size is 0.1-10mg.
9. according to the method described in claim 8, it is characterized by: the method includes the following steps:
0.1-10mg insect sample is added in extractant described in any one of 10-100 μ L claim 1-5,5-7 is ground
Second, vortex 2-3 seconds, it is centrifuged 2-3 seconds removal impurity, obtained supernatant i.e. insect sample DNA solution.
10. according to the method described in claim 8, it is characterized by: the method includes the following steps:
0.1-10mg insect sample is added in extractant described in any one of 10-100 μ L claim 1-5,5- is ground
7s stands 3-15s, obtains insect sample DNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811461792.7A CN109371013B (en) | 2018-12-02 | 2018-12-02 | Extracting agent for extracting DNA from insect material and application thereof |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243090A (en) * | 2013-05-31 | 2013-08-14 | 遵义医学院 | Method for extracting DNA (deoxyribonucleic acid) of insects |
| CN104928281A (en) * | 2015-05-29 | 2015-09-23 | 中国农业大学 | Method for improving DNA (Deoxyribonucleic Acid) extraction quality of cotton seed |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243090A (en) * | 2013-05-31 | 2013-08-14 | 遵义医学院 | Method for extracting DNA (deoxyribonucleic acid) of insects |
| CN104928281A (en) * | 2015-05-29 | 2015-09-23 | 中国农业大学 | Method for improving DNA (Deoxyribonucleic Acid) extraction quality of cotton seed |
Non-Patent Citations (2)
| Title |
|---|
| ZHANGUO XIN 等: "High-Throughput DNA Extraction Method Suitable for PCR", 《BIOTECHNIQUES》 * |
| 陈建军 等: "IC-RT -PCR检测葡萄卷叶病毒Ⅲ的研究", 《河南农业科学》 * |
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