CN105886495A - Method for extracting DNA (deoxyribonucleic acid) from feces of Chinese box turtles - Google Patents
Method for extracting DNA (deoxyribonucleic acid) from feces of Chinese box turtles Download PDFInfo
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- 241001194819 Cuora flavomarginata Species 0.000 title claims abstract description 46
- 210000003608 fece Anatomy 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 23
- 108020004414 DNA Proteins 0.000 title abstract description 18
- 102000053602 DNA Human genes 0.000 title abstract 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims abstract description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000006228 supernatant Substances 0.000 claims abstract description 20
- 239000000284 extract Substances 0.000 claims abstract description 16
- 239000000203 mixture Substances 0.000 claims abstract description 15
- 238000002156 mixing Methods 0.000 claims abstract description 14
- 239000002244 precipitate Substances 0.000 claims abstract description 13
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000001556 precipitation Methods 0.000 claims description 18
- 239000006166 lysate Substances 0.000 claims description 12
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 6
- 229960004756 ethanol Drugs 0.000 claims description 6
- 238000012545 processing Methods 0.000 claims description 4
- 238000007400 DNA extraction Methods 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 230000008569 process Effects 0.000 claims description 2
- 239000007788 liquid Substances 0.000 abstract description 8
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 3
- 108010067770 Endopeptidase K Proteins 0.000 abstract 2
- 230000009089 cytolysis Effects 0.000 abstract 2
- LSSUMOWDTKZHHT-UHFFFAOYSA-N N,n-diethyltryptamine Chemical class C1=CC=C2C(CCN(CC)CC)=CNC2=C1 LSSUMOWDTKZHHT-UHFFFAOYSA-N 0.000 abstract 1
- 238000001035 drying Methods 0.000 abstract 1
- 235000019441 ethanol Nutrition 0.000 abstract 1
- 238000002791 soaking Methods 0.000 abstract 1
- 238000005406 washing Methods 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 15
- 241001465754 Metazoa Species 0.000 description 6
- 241000894007 species Species 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 230000002550 fecal effect Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 108020005196 Mitochondrial DNA Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 230000027326 copulation Effects 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 2
- UELITFHSCLAHKR-UHFFFAOYSA-N acibenzolar-S-methyl Chemical compound CSC(=O)C1=CC=CC2=C1SN=N2 UELITFHSCLAHKR-UHFFFAOYSA-N 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
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- 238000011534 incubation Methods 0.000 description 2
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- 238000005070 sampling Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 1
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000270605 Chelonia Species 0.000 description 1
- 241000270693 Chinemys Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000032442 Geoemydidae Species 0.000 description 1
- 108091092878 Microsatellite Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 241000270666 Testudines Species 0.000 description 1
- 241000270708 Testudinidae Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- 230000017448 oviposition Effects 0.000 description 1
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- 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
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Abstract
The invention relates to a method for extracting DNA (deoxyribonucleic acid) from feces of Chinese box turtles, and belongs to the field of technologies for separating and purifying DNA. The method includes collecting the feces of the Chinese box turtles as samples, immediately filling a sterilized centrifugal tube with the feces and soaking the centrifugal tube in DETs (diethyltryptamines); adding dithiothreitol lysis solution into the centrifugal tube, uniformly mixing the dithiothreitol lysis solution and the feces with each other and then carrying out water bath treatment; quantitatively adding protease K (PK) into the centrifugal tube and allowing the centrifugal tube to stand still on ice; centrifuging the centrifugal tube, acquiring supernatant, filling another sterile centrifugal tube with the supernatant and discarding precipitates; adding phenol/chloroform/isoamyl alcohol mixed liquid into the supernatant and extracting the supernatant to obtain extract liquid; adding NaAc into the extract liquid, uniformly mixing the NaAc and the extract liquid with each other on a vortex mixer to obtain a first mixture, adding precooled absolute ethyl alcohol into the first mixture, sufficiently mixing the absolute ethyl alcohol and the first mixture with each other to obtain a second mixture and then allowing the second mixture to stand still; centrifuging the second mixture, discarding supernatant in a suction manner and drying precipitates at the room temperature; adding ethyl alcohol into the precipitates and washing the precipitates; airing the precipitates at the room temperature, adding TE into the precipitates when the precipitates tend to be transparent and dissolving the precipitates. The volume of the phenol/chloroform/isoamyl alcohol mixed liquid is equal to that of the corresponding supernatant. The method has the advantages that the method is applied to extracting the DNA of the Chinese box turtles and can be implemented conveniently and quickly, the Chinese box turtles are hardly injured, and the like.
Description
Technical field
The present invention relates to a kind of method that Cistoclemmys flavomarginata feces extracts DNA, belong to DNA separating and purifying technology
Field.
Background technology
Cistoclemmys flavomarginata (Cistoclemmys flavomarginata, Yellow-margined Box Turtle) is under the jurisdiction of
Chelonia (Testudinata), fresh water Testudinidae (Geoemydidae), box Chinemys (Cistoclemmys).Because of its carapace
Edge shield abdominal part yellow, eye socket has a golden yellow striped, therefore gains the name.Cistoclemmys flavomarginata is distributed mainly in China
The ground such as Zhejiang, Anhui, Fujian, Jiangsu, Henan, Guangxi, Guangdong, Taiwan, also there is distribution in Japan of neighbouring country.
Our province is mainly distributed on the ground such as Linan, Jiande, Anji, Long You, balcony, Xinchang.Owing to Cistoclemmys flavomarginata is Testudinis
Middle treasure, is senior tonic, and its medical value is higher, also has bigger ornamental value.In recent years,
Owing to artificial transition catches, wild Cistoclemmys flavomarginata resource and habitat thereof are by heavy damage, and wild quantity is drastically
Reduce.Within 1998, this population has been put into country rare species, within 2006, is classified as level in imminent danger by the United Nations and moves
Species group (IUCN, 2006).In addition to anthropic factor, due to wild Cistoclemmys flavomarginata poor growth, in addition,
Frequently occurring of global warming and badly freakish weather so that it is copulation environment, incubation condition etc. are with the suitableeest
Condition has had no small gap, so egg laying amount is few after sexual maturity, and rate of fertilization, incubation rate and survival rate all pole
Low, and its sex differentiation etc. is also created considerable influence, cause wild stocks endangered.
At present, the Germplasm Resource Investigation of Cistoclemmys flavomarginata wild to our province, and with close on province wild stocks relationship
Relation analysis is identified and is the most effectively carried out, and Cistoclemmys flavomarginata plan ecology chartering cost is carried out still being in the exploratory stage.
Existing several the breeding enterprises of our province start exploratory cultivation Cistoclemmys flavomarginata, but owing to Cistoclemmys flavomarginata growth cycle is long,
Pure lines and hybridization Testudinis are less in young Testudinis phase difference, and after cultivation 6-8, treasure Cistoclemmys flavomarginata character just can manifest, from
And create bigger cultivation risk.The sample being conventionally used to DNA extraction is mainly derived from blood or tissue
Block, but for wild animals on the brink of extinction, take blood and obtain piece of tissue all animal itself is had injury and likely
Bring disease to infect, its existence is had certain hidden danger.
Based on this, make the application.
Summary of the invention
In order to overcome existing Cistoclemmys flavomarginata identify existing for drawbacks described above, the application provide a kind of simple and easy to do,
Method easy to operate, that extract DNA without the in vitro Cistoclemmys flavomarginata feces dissected.
For achieving the above object, the technical scheme that the application takes is as follows:
A kind of Cistoclemmys flavomarginata feces extracts the method for DNA, comprises the steps: that (1) is by Cistoclemmys flavomarginata excrement
Just after as sample collecting, load in sterilized centrifuge tube immediately, with DETs (DMSO/EDTA/Tris
Saline solution) soak；(2) take the feces in above-mentioned immersion in centrifuge tube, add dithiothreitol, DTT (DTT)
Water bath processing after lysate, lysate and feces mixing；(3) after addition E.C. 3.4.21.64 (PK) quantitatively, vertical
I.e. put into and stand on ice；(4) centrifugal, take supernatant in another sterile centrifugation tube, abandon precipitation；In supernatant
Add isopyknic phenol/chloroform/isoamyl alcohol mixed liquor extracting；(5) add NaAc to mix on vortex mixer
After, add the dehydrated alcohol of pre-cooling, fully stand after mixing；(6) recentrifuge, inhales and abandons supernatant, heavy
Form sediment airing at room temperature；(7) add volumetric concentration 70% ethanol, relaunder precipitation；(8) room temperature is dried,
Add TE to dissolve when precipitation tends to transparent.
Further, as preferably:
In step (1), sample is from collecting the DNA extraction room temperature for step (2) and subsequent step thereof
Preserve 90 days～120 days.
In step (2), lysate is configured to: 100mmol/LTris-HCl, pH8,1.4mol/LNaCl,
20mmol/LEDTA, 0.4mol/L DTT.
In step (2), bath temperature is 40～60 DEG C, water bath time 1-4 hour.
In step (3), after processing 5～15 minutes at 50-55 DEG C after addition E.C. 3.4.21.64 is quantitative, Yu Bing
Upper standing 5～15 minutes.
In step (4), in phenol/chloroform/isoamyl alcohol mixed liquor, phenol: chloroform: isoamyl alcohol=25: 24: 1 (bodies
Long-pending ratio).
In step (5), NaAc concentration is 1.5～5mol/L, and addition is solution body after step (4) processes
Long-pending 0.1～0.3 times；The addition of dehydrated alcohol is to add after NaAc 1～3 times of liquor capacity；Mixing
After at-10～-30 DEG C stand 0.5～1 hour.
By the enforcement of technical scheme, evaluate wild Cistoclemmys flavomarginata germ plasm resource by collecting, use " row
Let out thing " sampling mode, this mode can utilize these samples in the case of not injuring and even must not seeing animal
Product extract animal DNA and go forward side by side performing PCR amplification, and the recycling hereditism such as mtDNA, PCR, molecule are raw
Thing learns a skill, and sets up Cistoclemmys flavomarginata kind matter Molecular Identification and analyzes method, it is achieved do not affecting wild stocks distribution
Cistoclemmys flavomarginata wild resource present situation is verified under premise, the Cistoclemmys flavomarginata kind matter method for identifying molecules set up, favorably
In verifying wild Cistoclemmys flavomarginata germ plasm resource, and by the life of wild Cistoclemmys flavomarginata, copulation, the bar such as breed
Part is investigated, and builds Cistoclemmys flavomarginata and intends ecological chartering cost system, thus carries out the analysis of bion hereditary information；
Finally it is effectively protected this Endangered species, for protection species diversity, also holding for Chinese medicine natural resources
Continue and offer approach is provided.
Accompanying drawing explanation
Fig. 1 is the agarose nucleic acid electrophoresis detection spectrogram of the application.
Detailed description of the invention
Embodiment 1
Fecal specimens loads in sterilized centrifuge tube the most immediately, with DETs (DMSO/EDTA/Tris
Saline solution) soak, room temperature keeps 90 days, stand-by.
1) take the above-mentioned feces of 100mg in 1mL centrifuge tube, add 1mL dithiothreitol, DTT (DTT) and split
Solving liquid, mix rear 40 DEG C of water-bath 4h, wherein lysate is configured to: 100mmol/LTris-HCl, pH=8,
1.4mol/LNaCl, 20mmol/LEDTA, 0.4mol/L DTT；
2) addition E.C. 3.4.21.64 (PK) is to 0.02mg, 55 DEG C, and 10min is immediately placed in 15min on ice.
3) 12000g is centrifuged 5min, takes supernatant in another aseptic 1.5mL centrifuge tube, abandons precipitation.Upwards
Addition equal-volume phenol in clear: chloroform: isoamyl alcohol is the mixture extracting of 25: 24: 1；
4) add the 1.5mol/LNaAc of 0.15 times of volume, vortex mixer adds after mixing 2 times of volumes
The dehydrated alcohol of (adding the volume calculated after salt) pre-cooling, fully-10 DEG C of placement 0.5h～1h after mixing；
5) 12000r/min is centrifuged 10min, inhales and abandons supernatant, precipitates airing at room temperature；
6) add 500 μ L70% ethanol and relaunder precipitation 2 times；
7) room temperature is dried, and adds 100 μ LTE when precipitation tends to transparent and dissolves.
Embodiment 2
Fecal specimens loads in sterilized centrifuge tube the most immediately, with DETs (DMSO/EDTA/Tris
Saline solution) soak, room temperature keeps 100 days, stand-by.
1) take the above-mentioned feces of 100mg in 1mL centrifuge tube, add 1mL dithiothreitol, DTT (DTT) and split
Solving liquid, mix rear 45 DEG C of water-bath 3h, wherein lysate is configured to: 100mmol/LTris-HCl, pH=8,
1.4mol/LNaCl, 20mmol/LEDTA, 0.4mol/L DTT；
2) addition E.C. 3.4.21.64 (PK) is to 0.02mg, 50 DEG C, and 15min is immediately placed in 5min on ice.
3) 12000g is centrifuged 5min, takes supernatant in another aseptic 1.5mL centrifuge tube, abandons precipitation.Upwards
Addition equal-volume phenol in clear: chloroform: isoamyl alcohol is the mixture extracting of 25: 24: 1；
4) add the 4mol/LNaAc of 0.2 times of volume, vortex mixer adds after mixing 2 times of volumes and (adds
Enter the volume calculated after salt) dehydrated alcohol of pre-cooling, fully after mixing-20 DEG C place 0.5h～1h；
5) 12000r/min is centrifuged 10min, inhales and abandons supernatant, precipitates airing at room temperature；
6) add 500 μ L70% ethanol and relaunder precipitation 2 times；
7) room temperature is dried, and adds 100 μ LTE when precipitation tends to transparent and dissolves.
Embodiment 3
Fecal specimens loads in sterilized centrifuge tube the most immediately, with DETs (DMSO/EDTA/Tris
Saline solution) soak.
1) take the feces of the above-mentioned immersion of 100mg in 1mL centrifuge tube, add 1mL dithiothreitol, DTT (DTT)
Lysate, lysate (100mmol/LTris-HCl, pH=8,1.4mol/LNaCl, 20mmol/LEDTA,
0.4mol/L DTT), mix rear 55 DEG C of water-bath 2h；
2) addition E.C. 3.4.21.64 (PK) is to 0.02mg, 50 DEG C, and 8min is immediately placed in 10min on ice.
3) 12000g is centrifuged 5min, takes supernatant in another aseptic 1.5mL centrifuge tube, abandons precipitation.Upwards
Addition equal-volume phenol in clear: chloroform: isoamyl alcohol is the mixture extracting of 25: 24: 1；
4) add the 3mol/LNaAc of 0.1 times of volume, vortex mixer adds after mixing 1.5 times of volumes
The dehydrated alcohol of (adding the volume calculated after salt) pre-cooling, fully-20 DEG C of placement 0.5h～1h after mixing；
5) 12000r/min is centrifuged 10min, inhales and abandons supernatant, precipitates airing at room temperature；
6) add 500 μ L70% ethanol and relaunder precipitation 2 times；
7) room temperature is dried, and adds 100 μ LTE when precipitation tends to transparent and dissolves.
Embodiment 4
Fecal specimens loads in sterilized centrifuge tube the most immediately, with DETs (DMSO/EDTA/Tris
Saline solution) soak, room temperature keeps 120 days, stand-by.
1) take the above-mentioned feces of 100mg in 1mL centrifuge tube, add 1mL dithiothreitol, DTT (DTT) and split
Solving liquid, mix rear 60 DEG C of water-bath 1h, wherein lysate is configured to: 100mmol/LTris-HCl, pH=8,
1.4mol/LNaCl, 20mmol/LEDTA, 0.4mol/L DTT；
2) addition E.C. 3.4.21.64 (PK) is to 0.02mg, 55 DEG C, and 5min is immediately placed in 10min on ice.
3) 12000g is centrifuged 5min, takes supernatant in another aseptic 1.5mL centrifuge tube, abandons precipitation.Upwards
Addition equal-volume phenol in clear: chloroform: isoamyl alcohol is the mixture extracting of 25: 24: 1；
4) add the 5mol/LNaAc of 0.3 times of volume, vortex mixer adds after mixing 3 times of volumes and (adds
Enter the volume calculated after salt) dehydrated alcohol of pre-cooling, fully after mixing-30 DEG C place 0.5h～1h；
5) 12000r/min is centrifuged 10min, inhales and abandons supernatant, precipitates airing at room temperature；
6) add 500 μ L70% ethanol and relaunder precipitation 2 times；
7) room temperature is dried, and adds 100 μ LTE when precipitation tends to transparent and dissolves.
The lysate of above-described embodiment is handled as follows:
A () PCR primer designs
Table 1 Cistoclemmys flavomarginata mitochondrial genome complete sequence amplimer
B () PCR expands
PCR amplification uses Japan to spin the high-fidelity enzyme KOD FX of (Shanghai) Bioisystech Co., Ltd.
Table 2PCR reactive component
| Reactive component | Volume (μ l) |
| Forward primer | 0.3 |
| Downstream primer | 0.3 |
| DNA profiling | 1 |
| KOD FX(1U/μl) | 0.1 |
| 2×PCR Buffer for KOD FX | 10 |
| 2mM dNTP | 1 |
| ddH2O | Up to 20 |
Table 3PCR response procedures
Mitochondrial gene PCR primer is carried out sepharose electrophoresis detection, and reclaims test kit (DNA with gel
GelExtractionKit, Takara) carry out the recovery of purpose segment；Microsatellite PCR product is carried out polypropylene
Amide electrophoresis detection；It is purified recovery with purification kit (E.Z.N.ACyclePureKit, Omega).Return
Product after receipts checks order, and this sequence can be used for comparison analysis in ncbi database, i.e. completes whole
The extraction of DNA and detection application.
Electrophoresis detection: 1% sepharose electrophoresis, in conjunction with Fig. 1, it can be seen that DNA band is obvious, and DNA adopts
Collection purity is higher.
By the enforcement of technical scheme, evaluate wild Cistoclemmys flavomarginata germ plasm resource by collecting, use " row
Let out thing " sampling mode, this mode can utilize these samples in the case of not injuring and even must not seeing animal
Product extract animal DNA and go forward side by side performing PCR amplification, and the recycling hereditism such as mtDNA, PCR, molecule are raw
Thing learns a skill, and sets up Cistoclemmys flavomarginata kind matter Molecular Identification and analyzes method, it is achieved do not affecting wild stocks distribution
Cistoclemmys flavomarginata wild resource present situation is verified under premise, the Cistoclemmys flavomarginata kind matter method for identifying molecules set up, favorably
In verifying wild Cistoclemmys flavomarginata germ plasm resource, and by the life of wild Cistoclemmys flavomarginata, copulation, the bar such as breed
Part is investigated, and builds Cistoclemmys flavomarginata and intends ecological chartering cost system, thus carries out the analysis of bion hereditary information；
Finally it is effectively protected this Endangered species, for protection species diversity, also holding for Chinese medicine natural resources
Continue and offer approach is provided.
Above content is that the preferred implementation combining the invention is entered one to what provided technical scheme made
Step describes in detail, it is impossible to assert that the invention is embodied as being confined to these explanations above-mentioned, for the present invention
For creating person of an ordinary skill in the technical field, without departing from the concept of the premise of the invention, also
Some simple deduction or replace can be made, all should be considered as belonging to the protection domain of the invention.
Claims (7)
1. the method that a Cistoclemmys flavomarginata feces extracts DNA, it is characterised in that comprise the steps:
(1) using Cistoclemmys flavomarginata feces as after sample collecting, load immediately in sterilized centrifuge tube, soak with DETs；
(2) take the feces in above-mentioned immersion in centrifuge tube, add lysate, lysate mix with feces after water bath processing；
(3), after addition E.C. 3.4.21.64 is quantitative, it is immediately placed in and stands on ice；
(4) centrifugal, take supernatant in another sterile centrifugation tube, abandon precipitation；Isopyknic phenol/chloroform/isoamyl alcohol mixed liquor extracting is added in supernatant；
(5) after addition NaAc mixes on vortex mixer, add the dehydrated alcohol of pre-cooling, fully stand after mixing；
(6) recentrifuge, inhales and abandons supernatant, precipitate airing at room temperature；
(7) add volumetric concentration 70% ethanol, relaunder precipitation；
(8) room temperature is dried, and adds TE when precipitation tends to transparent and dissolves.
A kind of Cistoclemmys flavomarginata feces the most as claimed in claim 1 extracts the method for DNA, it is characterised in that: in step (1), sample preserves 90 days～120 days from the DNA extraction room temperature collected for step (2) and subsequent step thereof.
A kind of Cistoclemmys flavomarginata feces the most as claimed in claim 1 extracts the method for DNA, it is characterised in that: in step (2), lysate is configured to: 100 mmol/LTris-HCl, pH8,1.4 mol/LNaCl, 20mmol/LEDTA, 0.4 mol/L DTT.
A kind of Cistoclemmys flavomarginata feces the most as claimed in claim 1 extracts the method for DNA, it is characterised in that: in step (2), bath temperature is 40～60 DEG C, water bath time 1-4 hour.
A kind of Cistoclemmys flavomarginata feces the most as claimed in claim 1 extracts the method for DNA, it is characterised in that: in step (3), after processing 5～15 minutes at 50-55 DEG C after addition E.C. 3.4.21.64 is quantitative, stand 5～15 minutes on ice.
A kind of Cistoclemmys flavomarginata feces the most as claimed in claim 1 extracts the method for DNA, it is characterised in that: in step (4), in phenol/chloroform/isoamyl alcohol mixed liquor, phenol: chloroform: isoamyl alcohol=25: 24: 1.
A kind of Cistoclemmys flavomarginata feces the most as claimed in claim 1 extracts the method for DNA, it is characterised in that: in step (5), NaAc concentration is 1.5～5mol/L, and addition is 0.1～0.3 times of liquor capacity after step (4) processes；The addition of dehydrated alcohol is to add after NaAc 1～3 times of liquor capacity；At-10～-20 DEG C, 0.5～1 hour is stood after mixing.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718728A (en) * | 2004-07-07 | 2006-01-11 | 李迪强 | Extraction method of Pu goa feaces DNA |
| CN1904066A (en) * | 2006-07-15 | 2007-01-31 | 安徽大学 | Extraction method of mammal faeces DNA |
| CN103146683A (en) * | 2013-02-19 | 2013-06-12 | 北京林业大学 | Method for extracting DNA from excrements of mammals and birds |
-
2016
- 2016-06-17 CN CN201610439317.4A patent/CN105886495B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718728A (en) * | 2004-07-07 | 2006-01-11 | 李迪强 | Extraction method of Pu goa feaces DNA |
| CN1904066A (en) * | 2006-07-15 | 2007-01-31 | 安徽大学 | Extraction method of mammal faeces DNA |
| CN103146683A (en) * | 2013-02-19 | 2013-06-12 | 北京林业大学 | Method for extracting DNA from excrements of mammals and birds |
Non-Patent Citations (2)
| Title |
|---|
| 申剑等: "三种粪便总DNA提取方法的比较", 《中国微生态学杂志》 * |
| 陶新等: "少量动物粪便细菌总DNA提取方法的研究", 《动物生产》 * |
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