CN106282164A - A kind of rapid extracting method of black porgy genomic DNA - Google Patents
A kind of rapid extracting method of black porgy genomic DNA Download PDFInfo
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- CN106282164A CN106282164A CN201610707121.9A CN201610707121A CN106282164A CN 106282164 A CN106282164 A CN 106282164A CN 201610707121 A CN201610707121 A CN 201610707121A CN 106282164 A CN106282164 A CN 106282164A
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- black porgy
- genomic dna
- extracting method
- rapid extracting
- porgy
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- 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
Abstract
The present invention relates to the rapid extracting method of a kind of black porgy genomic DNA, including: black porgy fin ray or muscle samples are ground or shred, adds Extraction buffer, concussion mixing, obtain black porgy tissue extract；Then place 20～40min in 65 DEG C, take out sample, cooling, stand or centrifuging and taking supernatant, obtain black porgy genomic DNA.The extraction step of the present invention is simple, quick, requires relatively low to experimental apparatus, and the extraction of genomic DNA can complete in 1h, and it is more convenient to extract compared to test kit, has a good application prospect.
Description
Technical field
The invention belongs to extracting genome DNA field, particularly to the rapid extracting method of a kind of black porgy genomic DNA.
Background technology
The large-scale application studied Fish along with molecular biology method, the extraction of genomic DNA has had become as one
One of kind the most frequently used technology, is the precondition and guarantee that successfully carries out of all kinds of research.
Phenol/chloroform extraction method is a kind of conventional DNA extraction method, is extracted by organic solvent, removes removing protein, polysaccharide,
The impurity such as phenols, it is common that the sample that will handle well, are separately added into cell pyrolysis liquid, E.C. 3.4.21.64, phenol and chloroform and isoamyl
The mixed liquor of alcohol progressively processes, and adds ethanol precipitation and can make separate nucleic acid out.Phenol can make protein denaturation,
Suppress the Degradation of DNase simultaneously.After adding phenol, owing to albumen is the most disconnected with DNA link button, protein molecular surface is contained again
Much polar group is similar to phenol mixes.Protein molecular is dissolved in phenol phase, and DNA is soluble in the aqueous phase.It is molten that chloroform can remove nucleic acid
Phenol in liquid, accelerates organic facies and stratified liquid.Isoamyl alcohol can reduce surface tension, reduces protein denaturation operating process
The bubble of middle generation, isoamyl alcohol contributes to split-phase simultaneously, makes the aqueous phase containing DNA of the upper strata after being centrifuged and the organic solvent phase of lower floor
Remain stable.
Phenol/chloroform extraction method is most common method during Fish genomes extracts, and the method technology is the most ripe, extracts effect
Fruit is preferable, but operates relatively complicated, the longest, and extraction reagent has toxicity more, easily damages experiment operator.
The cost using test kit to extract is the highest, is not suitable for a large amount of extractions of genomic DNA.The most many biotechnology handss
Section, such as the qualification of family in genetic breeding, population genetic diversity analysis, utilizes molecular marker to comment enhancement effect
Estimating, be required for a large amount of individuality and be analyzed or examination, extracting genome DNA workload is bigger.So, it is badly in need of invention a kind of fast
Speed, the DNA extraction method of low cost will provide convenient for these researchs.
The patent No. 201210031745.5 discloses a kind of grass carp genome DNA rapid extraction method, compared to the method,
PCR can be reacted, without SDS, EDTA etc., the material having a direct impact by agents useful for same of the present invention, and therefore, DNA extraction liquid can be without
The mode purification such as adsorption column are directly used in PCR reaction.Step is greatly simplified, and the operating time is shorter, and the requirement to equipment is lower, carries
Take cost the lowest, execute-in-place can be carried out in the place that experimental condition is the simplest and the crudest.
Summary of the invention
The technical problem to be solved is to provide the rapid extracting method of a kind of black porgy genomic DNA, the method
Extraction step is simple, quick, requires relatively low to experimental apparatus, and the extraction of genomic DNA can complete in 1h, compared to test kit
It is more convenient to extract.
A kind of rapid extracting method of the black porgy genomic DNA of the present invention, including:
Black porgy fin ray or muscle samples are ground or shred, adds Extraction buffer, concussion mixing, obtain black porgy tissue and carry
Take liquid；Then place 20～40min in 65 DEG C, take out sample, cooling, stand or centrifuging and taking supernatant, obtain black porgy genome
DNA；Wherein, Extraction buffer is the 100mM Tris-HCl of 1%TritonX-100, pH=8.0,300mM NaAc and 500mM
The mixed liquor of NaCl.
Described black porgy fin ray or muscle samples are fresh sample or dehydrated alcohol preservation sample.
Described Extraction buffer is 10～20 μ L:1 μ g with the ratio of black porgy fin ray or muscle samples.
Metal bath or thermostat water bath 65 DEG C is used to place 30min before described taking-up sample, between period or mixing.
The time of described standing is 3～5min.
Described centrifugal condition is that 13000r/min is centrifuged 3min.
Described black porgy genomic DNA can precipitate by adding 2 times of volume pre-cooling dehydrated alcohol, purer through 70% washing with alcohol
Change to meet higher requirement of experiment or preserve for long-term.
The patent No. 201210031745.5 discloses a kind of grass carp genome DNA rapid extraction method, compared to the method,
PCR can be reacted, without SDS, EDTA etc., the material having a direct impact by agents useful for same of the present invention, and therefore, DNA extraction liquid can be without
The mode purification such as adsorption column are directly used in PCR reaction.Step is greatly simplified, and the operating time is shorter, and the requirement to equipment is lower, carries
Take cost the lowest, execute-in-place can be carried out in the place that experimental condition is the simplest and the crudest.
Beneficial effect
(1) extraction step of the present invention is simple, quick, requires relatively low to experimental apparatus, and the extraction of genomic DNA can be in 1h
Completing, it is more convenient to extract compared to test kit；
(2) in the present invention, agents useful for same cost is extremely low, it is adaptable to the extensive extraction of black porgy genomic DNA；
(3) in the present invention, agents useful for same is safer, little to human injury, environmentally friendly；
(4) impact that in the present invention, PCR and enzyme action etc. are reacted by reagent is little, and DNA extraction liquid can not purified directly be carried out
Next step experimental implementation, further simplify experimental procedure, has a good application prospect.
Accompanying drawing explanation
Fig. 1 is the agarose gel electrophoresis figure of genomic DNA in embodiment 1；Wherein, M makes a living work biology DNA Marker
D；
Fig. 2 is the PCR expanding effect figure of genomic DNA in embodiment 2.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, people in the art
The present invention can be made various changes or modifications by member, and these equivalent form of values fall within the application appended claims equally and limited
Scope.
Embodiment 1
Black porgy dorsal rags sample extraction DNA is preserved from dehydrated alcohol, and for the PCR amplification of mitochondrial COⅠ gene.Real
Execute step as follows:
(1) clip 20 μ g soaked in absolute ethyl alcohol black porgy fin ray sample is in 1.5mL centrifuge tube, with glass grinding rod by sample
Fully grind at tube wall；
(2) 200 μ L Extraction buffer (1%TritonX-100+100mM Tris-HCl (pH=8.0)+300mM are added
NaAc+500mM NaCl), concussion 1min fully mixes；
(3) metal bath or thermostat water bath 65 DEG C place 30min, between period or mixing；
(4) taking out sample, cooling, in centrifuge, 13000r/min is centrifuged 3min；
(5) absorption supernatant is in another new pipe, expands for next step PCR.The black porgy genome extracted by this method
DNA, completely, the most satisfactory for result.(M work of making a living is raw as shown in Figure 1 for the black porgy genomic DNA agarose gel electrophoresis figure taken
Thing DNAMarker D).
Embodiment 2
(1) the fresh muscle samples of clip 20 μ g black porgy is in 1.5mL centrifuge tube, is filled at tube wall by sample with glass grinding rod
Divide and grind；
(2) 300 μ L Extraction buffer (1%TritonX-100+100mM Tris-HCl (pH=8.0)+300mM are added
NaAc+500mM NaCl), concussion 1min fully mixes, and obtains black porgy tissue extract；
(3) thermostat water bath 65 DEG C place 35min, between period or mixing；
(4) sample is taken out, cooling, stand 5min；
(5) supernatant is the black porgy genomic DNA obtained, and expands for next step PCR.
(6) with a pair black porgy mitochondrial COⅠ gene primer, said extracted DNA being carried out PCR amplification, primer sequence is:
Forward primer: 5 '-TACTCCTTACAGACCGAAAT-3 '；
Downstream primer: 5 '-TTATGAAGAAAGTGGCAGA-3 '；
Use 25 μ L amplification systems: 2.5 μ L 10 × PCR buffer, 0.5 μ L dNTP (10mmol/L), 1.5 μ L Mg2+
(25mmol/L), 2.5 μ L DNA extraction liquid, each 0.75 μ L of upstream and downstream primer (10 μm ol/L), 0.25 μ L Taq archaeal dna polymerase
(5U/ μ L), supplies volume with sterilizing DDW.Response procedures is: 94 DEG C of denaturations 5min, 94 DEG C of 45s, 52 DEG C of annealing 45s,
72 DEG C of 90s, 35 circulations, last 72 DEG C extend 10min.Amplified band is clear, without miscellaneous band, meets target size, permissible
Meet the needs of the analyses such as order-checking.Expanding effect is as shown in Figure 2.
Comparative example 1
Test kit extraction black porgy genomic DNA:
(1) 20-50mg tissue is cut into fine grained chippings, proceeds in the 1.5ml centrifuge tube equipped with 180 μ l Tissue lysates, with big
The piping and druming mixing of bore rifle head；
(2) Proteinase K Solution (20mg/ml) of 20 μ l, the fully mixing of vortex concussion at once are added；
(3) lysate being placed on 55 DEG C of water-baths 1-3 hour, period softly shakes several times；
(4) add 200 μ l and combine liquid, the fully mixing of vortex concussion at once, place 10min for 70 DEG C；
(5) 100 μ l isopropanols are added after cooling, the fully mixing of vortex concussion at once；
(6) by the rifle head withdraw mix of 1ml, feeding the mixture in an adsorption column, adsorption column puts into collecting pipe,
13000rpm is centrifuged 60s, outwells waste liquid in collecting pipe；
(7) adding 500 μ l mortifiers and remove liquid, 12000rpm is centrifuged 30s, discards waste liquid；
(8) adding 700 μ l rinsing liquids, 12000rpm is centrifuged 30s, discards waste liquid；
(9) being put back in sky collecting pipe by adsorption column, 13000rpm is centrifuged 2min, removes rinsing liquid as far as possible；
(10) take out adsorption column, put in clean centrifuge tube, add 100 μ l elution buffers in the middle part of adsorbed film
EB, room temperature placement 3-5 minute, 12000rpm is centrifuged 1 minute.In centrifuge tube, genomic DNA placement 4 DEG C is standby.
Comparative example 1 and comparative example 1 are it is found that test kit extraction step is loaded down with trivial details, and the reagent type of employing is many, technique
Cost is high, is not suitable for extensive operation.
Claims (6)
1. a rapid extracting method for black porgy genomic DNA, including:
Black porgy fin ray or muscle samples are ground or shred, adds Extraction buffer, concussion mixing, obtain black porgy tissue extraction
Liquid；Then place 20～40min in 65 DEG C, take out sample, cooling, stand or centrifuging and taking supernatant, obtain black porgy genomic DNA；
Wherein, Extraction buffer is the 100mM Tris-HCl of 1%TritonX-100, pH=8.0,300mM NaAc and 500mM
The mixed liquor of NaCl.
The rapid extracting method of a kind of black porgy genomic DNA the most according to claim 1, it is characterised in that described black porgy
Fin ray or muscle samples are fresh sample or dehydrated alcohol preservation sample.
The rapid extracting method of a kind of black porgy genomic DNA the most according to claim 1 and 2, it is characterised in that described in carry
The ratio taking buffer and black porgy fin ray or muscle samples is 10～20 μ L:1 μ g.
The rapid extracting method of a kind of black porgy genomic DNA the most according to claim 1, it is characterised in that described taking-up
Metal bath or thermostat water bath 65 DEG C is used to place 30min before sample, between period or mixing.
The rapid extracting method of a kind of black porgy genomic DNA the most according to claim 1, it is characterised in that described standing
Time be 5～10min.
The rapid extracting method of a kind of black porgy genomic DNA the most according to claim 1, it is characterised in that described centrifugal
Condition be 8000～13000r/min to be centrifuged 3min.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108226480A (en) * | 2017-12-22 | 2018-06-29 | 北京民海生物科技有限公司 | The method for measuring D- antigenic contents in aluminium salt absorbent-type DTap-IPV combined vaccines |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6037464A (en) * | 1995-09-27 | 2000-03-14 | Kim; Dong-Soo | Method for the extraction of genomic DNA from fish blood or sperm |
| CN103430936A (en) * | 2013-09-06 | 2013-12-11 | 中国水产科学研究院黑龙江水产研究所 | Preparation method of fish fin tissues for extracting genomic DNA (deoxyribonucleic acid) of fish |
| CN103911366A (en) * | 2014-03-19 | 2014-07-09 | 华南理工大学 | Genome DNA extraction method and its kit |
| CN105400775A (en) * | 2015-12-31 | 2016-03-16 | 中国长江三峡集团公司中华鲟研究所 | Method for harmlessly extracting Chinese sturgeon DNA |
-
2016
- 2016-08-23 CN CN201610707121.9A patent/CN106282164A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6037464A (en) * | 1995-09-27 | 2000-03-14 | Kim; Dong-Soo | Method for the extraction of genomic DNA from fish blood or sperm |
| CN103430936A (en) * | 2013-09-06 | 2013-12-11 | 中国水产科学研究院黑龙江水产研究所 | Preparation method of fish fin tissues for extracting genomic DNA (deoxyribonucleic acid) of fish |
| CN103911366A (en) * | 2014-03-19 | 2014-07-09 | 华南理工大学 | Genome DNA extraction method and its kit |
| CN105400775A (en) * | 2015-12-31 | 2016-03-16 | 中国长江三峡集团公司中华鲟研究所 | Method for harmlessly extracting Chinese sturgeon DNA |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108226480A (en) * | 2017-12-22 | 2018-06-29 | 北京民海生物科技有限公司 | The method for measuring D- antigenic contents in aluminium salt absorbent-type DTap-IPV combined vaccines |
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Application publication date: 20170104 |