CN107254466A - A kind of one-step method extracts the kit and method of DNA - Google Patents
A kind of one-step method extracts the kit and method of DNA Download PDFInfo
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- CN107254466A CN107254466A CN201710528302.XA CN201710528302A CN107254466A CN 107254466 A CN107254466 A CN 107254466A CN 201710528302 A CN201710528302 A CN 201710528302A CN 107254466 A CN107254466 A CN 107254466A
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- 239000000284 extract Substances 0.000 title claims abstract description 19
- 239000013612 plasmid Substances 0.000 claims abstract description 71
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- 239000012139 lysis buffer Substances 0.000 claims abstract description 28
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 24
- 235000010335 lysozyme Nutrition 0.000 claims abstract description 21
- 102000016943 Muramidase Human genes 0.000 claims abstract description 20
- 108010014251 Muramidase Proteins 0.000 claims abstract description 20
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims abstract description 20
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 claims abstract description 20
- 102000005891 Pancreatic ribonuclease Human genes 0.000 claims abstract description 20
- 229960000274 lysozyme Drugs 0.000 claims abstract description 20
- 239000004325 lysozyme Substances 0.000 claims abstract description 20
- 230000002101 lytic effect Effects 0.000 claims abstract description 20
- 239000012149 elution buffer Substances 0.000 claims abstract description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 30
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- 125000000400 lauroyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 8
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- 229960004198 guanidine Drugs 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 3
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- 108090000790 Enzymes Proteins 0.000 claims description 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- 229940088598 enzyme Drugs 0.000 claims description 2
- OTMSDBZUPAUEDD-UHFFFAOYSA-N Ethane Chemical compound CC OTMSDBZUPAUEDD-UHFFFAOYSA-N 0.000 claims 1
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- 239000011159 matrix material Substances 0.000 claims 1
- 125000000250 methylamino group Chemical group [H]N(*)C([H])([H])[H] 0.000 claims 1
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 abstract description 7
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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 discloses the kit and method that a kind of one-step method extracts DNA, belong to biological technical field.The technical scheme of use is that a kind of one-step method extracts the kit of DNA, including lytic reagent, rinsing liquid and elution buffer, and the lytic reagent includes lysozyme, RNase A and lysis buffer.Lysis buffer based on optimization, Synergistic lysozyme, RNase A rapid cleavage methods, realize one-step method cracking thalline i.e. upper prop, by plasmid extraction cycle time to 5min, and super spirial plasmid content is more than 90%, improve conventional efficient, high-quality plasmid provided for subsequent experimental, with it is efficient, quick, succinct the characteristics of.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of one-step method extracts the kit and method of DNA.
Background technology
DNA is the most common carrier of genetic engineering, is also increasingly becoming most important base in gene therapy research
Because of transmission system, it can be sent into recipient cell target DNA fragment by recombinant DNA technology and be bred and expressed.
The extraction and purifying of DNA are one of basic steps of molecular biology, are related to gene cloning, gene sequencing, nucleic acid
The fields such as vaccine, gene therapy and various genetic recombination, its efficiency extracted and quality are to follow-up molecular biology experiment (enzyme
Cut, PCR amplification and sequencing etc.) success or failure have close, direct relation.
Research finds that the plasmid extracted typically has supercoil, linear and open loop, and (DNA of double-stranded circular has part
Unwind to be formed) three kinds of conformations.The plasmid electrophoretic velocity of three kinds of conformations is supercoil matter when entering row agarose gel electrophoresis experiment
Grain>Shape material grain>Open circular plasmid, most fast with super spirial plasmid electrophoretic velocity, shape material grain is in centre, and open circular plasmid is most
Afterwards.Importantly, when with plasmid transfection eukaryotic, the transfection efficiency highest of super spirial plasmid, so in plasmid extraction
It is required that super spirial plasmid content will be more than 90%, this is also to the requirement as nucleic acid vaccine recombinant plasmid, super spirial plasmid
Content be judge plasmid extraction quality an important indicator.
The classical way with plasmid DNA purification is extracted from prokaryotes, step is followed successively by:Cellular lysate, plasmid separation
And purifying.Matching method, the current for example green skies of lot of domestic and international biological reagent company, raw work, Tiangeng etc., which are all developed, to be proposed
The kit of plasmid extraction, these kits carry out separation and Extraction plasmid with alkaline lysis combination purification column, pure with silica gel using plasmid
Change the suction-operated plasmid purification of post.Cellular lysate step therein, uses alkaline lysis, alkaline lysis plasmid DNA purification more
Technology is to be invented by Birnboim&Doly for 1979, and it, which is operated, includes three kinds of solution and handle, it is necessary to first step suspension thalline, the
Two step alkali and SDS denatured lysis and the 3rd step plasmid renaturation, then could go up centrifugal column, and extraction process generally requires 20 minutes left sides
The right time, experimental period is relatively long, and super spirial plasmid content is low, have impact on the experiment process of researcher;Germany
Eppendorf companies invention one-step method plasmid extraction method, based on lysozyme lysis bacterium, using containing lysozyme and detergent
Lysate cell lysis, pyrolysis product clarification is inviscid, directly can be combined with filter membrane after cracking, then by quickly centrifuging
Clean and afford the DNA of supercoil.It is quick and reproducible although the extracting method is convenient, it is not suitable for low
The extraction of plasmid and the extraction of rich medium culture escherichia coli plasmid are copied, and escherichia coli plasmid is conventional at present
One of plasmid, therefore, the application of this method have larger limitation.
It can be seen that, the method that plasmid is extracted at present is product, there is many many deficiencies, such as extraction efficiency is low, the cycle
It is long, influence experiment process;Limitation is stronger, without universality etc., and research and development universality is good, efficient, reliable extract is tried
Agent and method are significant to plasmid extraction, genetic engineering.
The content of the invention
It is existing to solve it is an object of the invention to provide the kit and method that a kind of one-step method extracts DNA
The technical problem that super spirial plasmid content is low, cost is high, limitation is big in extraction of plasmid DNA length experimental period, extract.This hair
It is bright to be based on lysozyme, RNase A methods, and using unique rapid cleavage buffer solution, one-step method cracking thalline i.e. upper prop is realized,
By plasmid extraction cycle time to 5min, and super spirial plasmid content improves conventional efficient more than 90%, is subsequent experimental
There is provided high-quality plasmid, with it is efficient, quick, succinct the characteristics of.
To achieve the above object, the technical solution adopted by the present invention is that a kind of one-step method extracts the kit of DNA,
Including lytic reagent, rinsing liquid and elution buffer, the lytic reagent includes lysozyme, RNase A and lysis buffer.
Further, the composition of the lysis buffer is:Per 100mL include lauroyl CAB 5~
10g, 1~2.5g of calcium chloride, 1~5g of dimethyl sulfoxide (DMSO), 1~2.5g of glycerine, 3- [3- (courage amido propyl) dimethylamino] third sulphur
Sour 1~2.5g of inner salt, three (methylol) aminomethane hydrochloride 1-2.5g, 1~2.5g of trishydroxymethylaminomethane, guanidine hydrochloride 1
~3.5g, 1~2.5g of guanidinium isothiocyanate, urea 5-10g, 1~2.5g of disodium ethylene diamine tetraacetate, remaining is water.
It is preferred that, the composition of the lysis buffer is:Include lauroyl CAB 10g, chlorination per 100mL
Calcium 1.1g, dimethyl sulfoxide (DMSO) 2.5g, glycerine 1g, 3- [3- (courage amido propyl) dimethylamino] propane sulfonic acid inner salt 1.25g, three (hydroxyls
Methyl) aminomethane hydrochloride 1.36g, trishydroxymethylaminomethane 1.65g, guanidine hydrochloride 2.39g, guanidinium isothiocyanate 1.18g, urine
Plain 6g, disodium ethylene diamine tetraacetate 1.86g, remaining is water.
Further, the lysis buffer is preceding according to lysis buffer in use:Lysozyme freeze-dried powder:10mg/mL's
RNase A are 100mL:250:350mg:1.5-2.5mL ratio mixing, forms lytic reagent.
It is preferred that, the lysis buffer:Lysozyme freeze-dried powder:10mg/mL RNase A are 100mL:300mg:2mL.
It is furthermore preferred that also including silicon substrate plasma membrane adsorption column in the kit.
The present invention also provides a kind of method that one-step method extracts DNA, the described method comprises the following steps:
1. the amplification of plasmid:The Escherichia coli bacteria liquid of 1~2mL incubated overnights is taken to be centrifuged with 10000-13000r/min
1min, abandons supernatant, obtains bacterial sediment thing;
2. cellular lysate:Add and wrapped in the lytic reagent of 400-600 μ L precoolings, the lytic reagent into bacterial sediment thing
Lysozyme, RNase A and lysis buffer are included, vortex oscillation 20-50s to bacterial sediment thing is completely dissolved, and obtains cellular lysate thing;
3. the purifying of DNA.
Further, the composition of the step 2. middle lysis buffer is:Include lauroyl propyl group beet per 100mL
5~10g of alkali, 1~2.5g of calcium chloride, 1~5g of dimethyl sulfoxide (DMSO), 1~2.5g of glycerine, 3- [3- (courage amido propyl) dimethylamino]
1~2.5g of propane sulfonic acid inner salt, three (methylol) aminomethane hydrochloride 1-2.5g, 1~2.5g of trishydroxymethylaminomethane, hydrochloric acid
1~3.5g of guanidine, 1~2.5g of guanidinium isothiocyanate, urea 5-10g, 1~2.5g of disodium ethylene diamine tetraacetate, remaining is water.
Further, the compound method of the step 2. lytic reagent is:Using preceding according to lysis buffer:Lysozyme freezes
Dry powder:10mg/mL RNase A are 100mL:250:350mg:1.5-2.5mL ratio well mixed formed.
Further, the purification process of the step 3. DNA is:Cellular lysate thing is transferred to silicon substrate plasma membrane adsorption column
In, with 10000-13000r/min centrifugation absorption 20s-1min, abandon waste liquid;200-400 μ are added into silicon substrate plasma membrane adsorption column again
L rinsing liquids, centrifuge 20-50s with 10000-13000r/min desalinations, abandon waste liquid;50~100 μ L elution buffers are added,
10000-13000r/min elution centrifugation 20-50s, obtain plasmid DNA solution.
There is provided the kit and method that a kind of one-step method extracts DNA in above-mentioned technical proposal, kit and method are
DNA is extracted based on lysozyme, RNase A and lysis buffer, it is slow that kit includes lytic reagent, rinsing liquid and elution
Fliud flushing, it is critical that described lytic reagent includes lysozyme, RNase A and lysis buffer.Lysozyme at room temperature may be used
The β between -acetylmuramic acid and NAG in partial destruction Bacillus coli cells wall-Isosorbide-5-Nitrae glycosidic bond, RNase
A can remove major part RNA, with reference to unique lysis buffer of the invention, on the one hand ensure that lysozyme and RNase A are active
Give full play to, improve lysis efficiency, DNA can on the other hand be sufficiently separated and quick from Escherichia coli with protein
Discharged in cell, while the joint efficiency of DNA and centrifugal adsorbing column can also be improved, lysozyme, RNase A and cracking
After buffer solution mixing, under the synergy of lysis buffer, whole extraction of plasmid DNA process is shortened to by traditional 20min
5min or so, the time for the person that greatlys save experimental study, improves conventional efficient, while supercoiled plasmid DNA content exists
More than 90%, it is that the success of subsequent experimental improves Reliable guarantee.
The inventive method has the following advantages that:(1) the unique lysis buffer energy Synergistic lysozyme of the present invention,
RNase A, realize rapidly and efficiently separation quality grain and albumen, and are discharged from Escherichia coli, while fracture cell walls, are realized efficient
The removal of impurity quickly is gone, lysis efficiency is substantially increased, improves extraction efficiency.(2) whole extraction of plasmid DNA process only 5min is left
The right side, the time for the person that greatlys save experimental study, improves conventional efficient, while supercoiled plasmid DNA content is more than 90%,
Reliable guarantee is improved for the success of subsequent experimental.
Brief description of the drawings
Swimming lane 1 is DNA point in the electrophoresis detection figure that Fig. 1 is extraction high copy number plasmid pBS in embodiment 1, figure
Sub- amount standard (Marker IV), swimming lane 2-5 is the DNA purified using the inventive method.
Fig. 2 is deoxyribose core to extract swimming lane 1 in the electrophoresis detection figure of low-copy pBR322 plasmid, figure in embodiment 2
Acid molecule amount standard (Marker IV), swimming lane 2-5 is the DNA purified using the inventive method.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the reagent that is related in the scope of the present invention, embodiment,
Unless otherwise specified, commercially obtain, involved operation, be routine operation unless otherwise specified.
The preparation of experiment reagent:
(1) preparation of lysis buffer:
Weigh or measure lauroyl 5~10g of CAB, 1~2.5g of calcium chloride, it is 1~5mL of DMSO, sweet
1~2.5mL of oil, 3- [3- (courage amido propyl) dimethylamino] 1~2.5g of propane sulfonic acid inner salt, three (methylol) aminomethane hydrochloric acid
1~2.5g of salt, 1~2.5g of trishydroxymethylaminomethane, 1~2.5g of guanidine hydrochloride, 1~2.5g of guanidinium isothiocyanate, 5~10g of urea
100mL is settled to 1~2.5g of disodium ethylene diamine tetraacetate, plus deionized water, is stirred, as experiment cracking buffering
Liquid.
Exemplary or preferred, above-mentioned lysis buffer composition is:Include lauroyl CAB per 100mL
10g, calcium chloride 1.1g, dimethyl sulfoxide (DMSO) 2.5g, glycerine 1g, 3- [3- (courage amido propyl) dimethylamino] propane sulfonic acid inner salt
1.25g, three (methylol) aminomethane hydrochloride 1.36g, trishydroxymethylaminomethane 1.65g, guanidine hydrochloride 2.39g, different sulphur cyanogen
Sour guanidine 1.18g, urea 6g, disodium ethylene diamine tetraacetate 1.86g, remaining is water.In the composition of lysis buffer, lauroyl third
Base glycine betaine and calcium chloride can increase the permeability of bacterial cell, make plasmid be easier to discharge from bacterium.Dimethyl is sub-
Sulfone and glycerine can play certain protective role to Bacillus coli cells, reduce the genome release confrontation caused by cell rupture
The interference that grain is extracted.3- [3- (courage amido propyl) dimethylamino] propane sulfonic acid inner salt is a kind of non denatured amphion detergent,
Protein cleavage liquid, is mainly used in dissolving memebrane protein, plasmid can be promoted to discharge.Three (methylol) aminomethane hydrochlorides
Constituted with trishydroxymethylaminomethane and crack buffer system used, low-concentration hcl guanidine, guanidinium isothiocyanate and urea are chaotropic agent
And strong denaturant, joint carries out Bacillus coli cells rapid cleavage, while being that nucleic acid and adsorbed film provide conjugation condition.Ethylenediamine
Tetraacethyl disodium can be with chela and bivalent metal ion, so as to suppress nuclease pair and DNA degraded.
Lysis buffer is adding 300mg lysozymes freeze-dried powder and 2 milliliters of 10mg/mL RNase A using preceding, and formation is split
Solve reagent.
(2) rinsing liquid is constituted:Final concentration of 20mM Tris and 200mM the NaCl aqueous solution (pH8.5);
(3) elution buffer is constituted:10mM Tris (pH8) buffer solution.
Embodiment 1 extracts high copy number plasmid pBS from culture of Escherichia coli
1. the amplification of plasmid:Take 1.5mL with the Escherichia coli bacteria liquid of LB culture medium incubated overnights, centrifuged with 12000r/min
1min, abandons supernatant, obtains bacterial sediment thing;
2. cellular lysate:The lytic reagent of 500 μ L precoolings is added into bacterial sediment thing, vortex oscillation 30s is heavy to thalline
Starch is completely dissolved, and obtains cellular lysate thing;
3. the purifying of DNA:By step 2. in cellular lysate thing room temperature place 2 minutes after be transferred to silicon substrate plasma membrane absorption
In post, with 12000r/min centrifugation absorption 30s, waste liquid is abandoned;300 μ L rinsing liquids are added into silicon substrate plasma membrane adsorption column again, with
12000r/min desalinations centrifuge 30s, abandon waste liquid;80 μ L elution buffers are finally added into silicon substrate plasma membrane adsorption column again,
12000r/min elution centrifugation 30s, obtain plasmid DNA solution.
Resulting DNA is subjected to detected through gel electrophoresis, accompanying drawing 1 is as a result seen, the wherein corresponding sample of swimming lane 1 is de-
Oxygen ribonucleic acid molecular weight standard (Marker IV), the corresponding samples of swimming lane 2-5 are above-mentioned kit and method with the present invention
The DNA of extraction, from result figure, the DNA content of the invention extracted is high, purity is high, free from admixture, is most worth noting
, total process that method of the invention and kit extract high copy number plasmid pBS from culture of Escherichia coli only takes
5min, greatlys save the time of experimenter, improves conventional efficient.
Embodiment 2 extracts high copy number plasmid pBS from culture of Escherichia coli
1. the amplification of plasmid:1.5mL is taken with the Escherichia coli bacteria liquid of MUG medium culture base incubated overnights, with 12000r/
Min centrifuges 1min, abandons supernatant, obtains bacterial sediment thing;
2. cellular lysate:The lytic reagent of 500 μ L precoolings is added into bacterial sediment thing, vortex oscillation 30s is heavy to thalline
Starch is completely dissolved, and obtains cellular lysate thing;
3. the purifying of DNA:By step 2. in cellular lysate thing room temperature place 2 minutes after be transferred to silicon substrate plasma membrane absorption
In post, with 12000r/min centrifugation absorption 30s, waste liquid is abandoned;300 μ L rinsing liquids are added into silicon substrate plasma membrane adsorption column again, with
12000r/min desalinations centrifuge 30s, abandon waste liquid;80 μ L elution buffers are finally added into silicon substrate plasma membrane adsorption column again,
12000r/min elution centrifugation 30s, obtain plasmid DNA solution.
Resulting DNA is subjected to detected through gel electrophoresis, as a result found, the DNA content height of the invention extracted,
Free from admixture, what 5min can be rapidly and efficiently extracts high copy number plasmid pBS, conventional efficient from culture of Escherichia coli.
Embodiment 3 extracts low-copy pBR322 plasmid from culture of Escherichia coli
1. the amplification of plasmid:Take 1mL with the Escherichia coli bacteria liquid of LB culture medium incubated overnights, centrifuged with 12000r/min
1min, abandons supernatant, obtains bacterial sediment thing;
2. cellular lysate:The lytic reagent of 500 μ L precoolings is added into bacterial sediment thing, vortex oscillation 30s is heavy to thalline
Starch is completely dissolved, and obtains cellular lysate thing;
3. the purifying of DNA:By step 2. in cellular lysate thing room temperature place 2 minutes after be transferred to silicon substrate plasma membrane absorption
In post, with 12000r/min centrifugation absorption 30s, waste liquid is abandoned;300 μ L rinsing liquids are added into silicon substrate plasma membrane adsorption column again, with
12000r/min desalinations centrifuge 30s, abandon waste liquid;80 μ L elution buffers are finally added into silicon substrate plasma membrane adsorption column again,
12000r/min elution centrifugation 30s, obtain plasmid DNA solution.
DNA obtained by embodiment 3 is subjected to detected through gel electrophoresis, accompanying drawing 2 is as a result seen, wherein swimming lane 1 is corresponding
Sample is DNA molecule amount standard (Marker IV), and the corresponding samples of swimming lane 2-5 are embodiment 3 with the examination of the present invention
The DNA that agent box and method are extracted, it is seen then that the DNA content of the invention extracted is high, free from admixture, from Escherichia coli culture
Total process that low-copy pBR322 plasmid is extracted in thing only takes 5min, greatlys save the time of experimenter, improves experiment
Efficiency.
Embodiment 4 extracts low-copy pBR322 plasmid from culture of Escherichia coli
1. the amplification of plasmid:Take 1mL with the Escherichia coli bacteria liquid of MUG culture medium incubated overnights, centrifuged with 12000r/min
1min, abandons supernatant, obtains bacterial sediment thing;
2. cellular lysate:The lytic reagent of 500 μ L precoolings is added into bacterial sediment thing, vortex oscillation 30s is heavy to thalline
Starch is completely dissolved, and obtains cellular lysate thing;
3. the purifying of DNA:By step 2. in cellular lysate thing room temperature place 2 minutes after be transferred to silicon substrate plasma membrane absorption
In post, with 12000r/min centrifugation absorption 30s, waste liquid is abandoned;300 μ L rinsing liquids are added into silicon substrate plasma membrane adsorption column again, with
12000r/min desalinations centrifuge 30s, abandon waste liquid;80 μ L elution buffers are finally added into silicon substrate plasma membrane adsorption column again,
12000r/min elution centrifugation 30s, obtain plasmid DNA solution.
DNA obtained by embodiment 4 is subjected to detected through gel electrophoresis, the DNA that the present invention is extracted as a result is found
Content is high, free from admixture.
The present invention can be used not only for the extraction of high copy and low-copy plasmid, be also applied for different culture media culture large intestine bar
The extraction of bacteria plasmid, the success rate for extracting plasmid improves 25-50% than current conventional method, it is often more important that greatly save reality
The time of researcher is tested, conventional efficient is improved.The DNA purity of purifying is greatly improved, and is applicable to various routine operations,
Including digestion, PCR, sequencing, connection, conversion, library screening, In Vitro Translation, transfect some conventional passage cells etc..The present invention
Compared with German Eppendorf companies one-step method plasmid extraction, by optimizing rapid cleavage buffer solution, Escherichia coli are added thin
The permeability of born of the same parents, makes plasmid be more easy to release, especially low-copy plasmid.Rapid cleavage buffer solution adds plasmid and absorption simultaneously
The joint efficiency of film, so that low-copy plasmid yield is improved, and purity is higher.
Although above with general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of one-step method extracts the kit of DNA, including lytic reagent, rinsing liquid and elution buffer, its feature exists
In the lytic reagent includes lysozyme, RNase A and lysis buffer.
2. kit according to claim 1, it is characterised in that the composition of the lysis buffer is:Wrapped in per 100mL
Include 5~10g of lauroyl CAB, 1~2.5g of calcium chloride, 1~5g of dimethyl sulfoxide (DMSO), 1~2.5g of glycerine, 3- [3- (courages
Amido propyl) dimethylamino] 1~2.5g of propane sulfonic acid inner salt, three (methylol) aminomethane hydrochloride 1-2.5g, trihydroxy methyl ammonia
1~2.5g of methylmethane, 1~3.5g of guanidine hydrochloride, 1~2.5g of guanidinium isothiocyanate, urea 5-10g, disodium ethylene diamine tetraacetate 1~
2.5g, remaining is water.
3. kit according to claim 1, it is characterised in that the composition of the lysis buffer is:Wrapped in per 100mL
Include lauroyl CAB 10g, calcium chloride 1.1g, dimethyl sulfoxide (DMSO) 2.5g, glycerine 1g, 3- [3- (courage amido propyl) two
Methylamino] propane sulfonic acid inner salt 1.25g, three (methylol) aminomethane hydrochloride 1.36g, trishydroxymethylaminomethane 1.65g, salt
Sour guanidine 2.39g, guanidinium isothiocyanate 1.18g, urea 6g, disodium ethylene diamine tetraacetate 1.86g, remaining is water.
4. the kit according to claim 1 or 2 or 3, it is characterised in that the lysis buffer using preceding according to splitting
Solve buffer solution:Lysozyme freeze-dried powder:10mg/mL RNase A are 100mL:250:350mg:1.5-2.5mL ratio mixing,
Form lytic reagent.
5. kit according to claim 4, it is characterised in that the lysis buffer:Lysozyme freeze-dried powder:10mg/
ML RNase A are 100mL:300mg:2mL.
6. the kit according to claim 1 or 2 or 3 or 5, it is characterised in that also include silicon matrix in the kit
Film adsorption column.
7. a kind of method that one-step method extracts DNA, it is characterised in that the described method comprises the following steps:
1. the amplification of plasmid:Take the Escherichia coli bacteria liquid of 1~2mL incubated overnights to centrifuge 1min with 10000-13000r/min, abandon
Supernatant, obtains bacterial sediment thing;
2. cellular lysate:The lytic reagent of 400-600 μ L precoolings is added into bacterial sediment thing, the lytic reagent includes molten
Bacterium enzyme, RNase A and lysis buffer, vortex oscillation 20-50s to bacterial sediment thing are completely dissolved, and obtain cellular lysate thing;
3. the purifying of DNA.
8. method according to claim 7, it is characterised in that the composition of the step 2. middle lysis buffer is:Often
100mL include 5~10g of lauroyl CAB, 1~2.5g of calcium chloride, 1~5g of dimethyl sulfoxide (DMSO), glycerine 1~
2.5g, 3- [3- (courage amido propyl) dimethylamino] 1~2.5g of propane sulfonic acid inner salt, three (methylol) aminomethane hydrochloride 1-
2.5g, 1~2.5g of trishydroxymethylaminomethane, 1~3.5g of guanidine hydrochloride, 1~2.5g of guanidinium isothiocyanate, urea 5-10g, ethylenediamine
1~2.5g of tetraacethyl disodium, remaining is water.
9. the method according to claim 7 or 8, it is characterised in that the compound method of the step 2. lytic reagent is:Make
With preceding according to lysis buffer:Lysozyme freeze-dried powder:10mg/mL RNase A are 100mL:250:350mg:1.5-2.5mL
Ratio is well mixed to be formed.
10. the method according to claim 7 or 8, it is characterised in that the purification process of the step 3. DNA is:Will
Cellular lysate thing is transferred in silicon substrate plasma membrane adsorption column, with 10000-13000r/min centrifugation absorption 20s-1min, abandons waste liquid;Again to
200-400 μ L rinsing liquids are added in silicon substrate plasma membrane adsorption column, 20-50s is centrifuged with 10000-13000r/min desalinations, waste liquid is abandoned;
50~100 μ L elution buffers are added, 10000-13000r/min elution centrifugation 20-50s obtain plasmid DNA solution.
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CN108866043A (en) * | 2018-07-17 | 2018-11-23 | 成都医学院 | A pretreatment kit and method for bacterial plasmid extraction |
CN109468311A (en) * | 2018-11-27 | 2019-03-15 | 河南普诺易生物制品研究院有限公司 | Bacterial lysate for extracting plasmid DNA, kit and method for extracting plasmid DNA |
CN111235226A (en) * | 2020-03-30 | 2020-06-05 | 广州达正生物科技有限公司 | Method for separating and purifying pathogenic microorganism DNA |
CN114657231A (en) * | 2022-04-27 | 2022-06-24 | 珠海宝锐生物科技有限公司 | Rapid DNA extraction kit with magnetic beads for fluorescent quantitative PCR detection and extraction method thereof |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108866043A (en) * | 2018-07-17 | 2018-11-23 | 成都医学院 | A pretreatment kit and method for bacterial plasmid extraction |
CN108866043B (en) * | 2018-07-17 | 2022-02-08 | 成都医学院 | A kind of pretreatment kit and method for bacterial plasmid extraction |
CN109468311A (en) * | 2018-11-27 | 2019-03-15 | 河南普诺易生物制品研究院有限公司 | Bacterial lysate for extracting plasmid DNA, kit and method for extracting plasmid DNA |
CN109468311B (en) * | 2018-11-27 | 2021-08-24 | 河南普诺易生物制品研究院有限公司 | Bacterial lysate for extracting plasmid DNA, kit and method for extracting plasmid DNA |
CN111235226A (en) * | 2020-03-30 | 2020-06-05 | 广州达正生物科技有限公司 | Method for separating and purifying pathogenic microorganism DNA |
CN114657231A (en) * | 2022-04-27 | 2022-06-24 | 珠海宝锐生物科技有限公司 | Rapid DNA extraction kit with magnetic beads for fluorescent quantitative PCR detection and extraction method thereof |
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