CN108410862A - The store method and kit of long-term gene group DNA - Google Patents
The store method and kit of long-term gene group DNA Download PDFInfo
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Abstract
The present invention provides the store methods and kit of long-term gene group DNA, and specifically the long-term preservation method of genomic DNA provided by the invention includes step：Solid phase carrier adsorbs target gene, washing removal impurity, dry removal liquid component, to realize the long-term preservation to genomic DNA.Genomic DNA is preserved using the method for the present invention, store method is expected effective holding time up to century-old for a long time.Solid phase material absorption is used to preserve genome DNA according to the method for the present invention, the integrality, physicochemical property and its biological nature of genomic DNA structure are not interfered with, and the genetic diversity of target gene can be kept, it is contemplated that more than effective holding time reachable a century.
Description
Technical field
The invention belongs to biotechnologies, specifically, the present invention relates to a kind of store methods of long-term gene group DNA
And kit.
Background technology
With the maturation of genome sequencing technology, Nature in 2009 has delivered two papers, wherein one to lung cancer
Cell and Skin Cell have carried out complete sequence determination, and finding lung carcinoma cell through sequence alignment, there are 22910 mutation；An another piece is right
Melanoma cells have carried out complete sequence determination with lymphocyte, and finding melanoma cells through sequence alignment, there are 33345
It is mutated (A small-cell lung cancer genome with complex signatures of tobacco
exposure.Nature.2010Jan 14；463(7278)；A comprehensive catalogue of somatic
mutations from a human cancer genome.Nature.2010Jan 14；463(7278)).More and more
Research has shown that a variety of diseases especially tumour is all to change group in turn since somatic mutation changes the performance of cell
The performance with organ is knitted, leads to disease symptoms occur.Therefore, the mutation of body cell is monitored, to treating and preventing disease
It is of great significance.
Although the development of genome sequencing technology is very fast, testing cost is also gradually reducing, at this stage complete
Gene order-checking expense is still very high, far beyond general population can tolerance range.Moreover, grinding with gene field
Study carefully gradually deeply, more and more gene alteration phenomenons be found and prove it is related to disease, such as DNA methylation modify, DNA
Skeleton sulfonylization modification etc..These progress show the expection before the remote superorder of the complexity of gene, existing technological means
It is difficult to which the variation to full-length genome level is comprehensively detected.
A solution is to be saved cdna sample with lower cost at present, by gene when needing future
Sample taking-up is detected.There is prediction to show that progress gene sequencing when baby due in 2025 conventional detection project will be become
One of.Therefore, with the progress of technique of gene detection, it is contemplated that in the near future, the expense of genetic test will significantly drop
The range that can be born as low as the whole people.
Currently, there are many preservation service that the Gene conservation company of profession carries out personal cdna sample, the genes of preservation
Group DNA can maintain the stabilization of its physicochemical property and biological activity for a long time.In existing technology, to keep the complete of gene structure
The stability of whole property and physicochemical property is required for greatly the genomic DNA sample for obtaining separation to be stored under condition of ultralow temperature, base
Because of preservation condition harshness, thus cost is higher.Therefore, those skilled in the art are dedicated to exploitation can preserve gene at normal temperatures
Technology, to further decrease the cost of Gene conservation, at the same ensure genomic DNA structure integrality and physicochemical property it is steady
It is qualitative, the loss to avoid genomic information or variation.
Invention content
The purpose of the present invention is to provide a kind of long-term preservation method of genomic DNA, kit and its applications.
In the first aspect of the present invention, a kind of long-term preservation method of genomic DNA is provided, the method includes steps
Suddenly：
(1) solid phase carrier adsorbs
Genomic DNA to be saved is provided, the genomic DNA is added in solid phase adsorption solution to mix well and is mixed
Close liquid；Wherein, the solid phase adsorption solution includes solid phase adsorption carrier, PEG and NaCl；
(2) it washs
The mixed liquor obtained in centrifugation step (1) removes supernatant；Organic solvent washing precipitation is added, is removed after centrifugation
Organic solvent mixing is added in supernatant；
(3) dry
The liquid that step (2) obtains is transferred to and is preserved in pipe, dry removal liquid component, to realize to genome
The long-term preservation of DNA.
In another preferred example, the solid phase adsorption carrier is nano silica microsphere.
In another preferred example, a diameter of 5~100nm of the silicon dioxide microsphere；Preferably 10~50nm；More preferably
About 20nm.
In another preferred example, the PEG is PEG8000.
In another preferred example, the solid phase adsorption pH value of solution ＜ 7.
In another preferred example, the solid phase adsorption pH value of solution is 4.5~6.5；Preferably 5.0~6.0；More preferably
5.5。
In another preferred example, the content of solid phase adsorption carrier is 0.05g/ml to 0.5g/ in the solid phase adsorption solution
ml；Preferably 0.1g/ml to 0.2g/ml.
In another preferred example, the content of PEG is 5%~40% (W/V) in the solid phase adsorption solution；Preferably 15%
~20% (W/V).
In another preferred example, the content of NaCl is 0.5~3M in the solid phase adsorption solution；Preferably 1~2M.
In another preferred example, the solid phase adsorption solution further includes Mg2+And Co2+, and Mg2+Molar concentration and Co2+It rubs
Your concentration ratio is 1~3:1；Preferably 3:2.
In another preferred example, Mg in the solid phase adsorption solution2+A concentration of 0.01~0.05mM；Preferably 0.03mM.
In another preferred example, Co in the solid phase adsorption solution2+A concentration of 0.005~0.03mM；Preferably 0.02mM.
In another preferred example, the solid phase adsorption solution includes：
Silicon dioxide microsphere 0.1g/ml, PEG80005%-40% (W/V) and NaCl 0.5-3M.
In another preferred example, further include in the solid phase adsorption solution：MgCl20.01~0.05mM and CoCl20.005
~0.03mM.
In another preferred example, in the step (3), long term shelf-life is 5 years or more；Preferably 10 years or more；More
Preferably 50 years or more；Most preferably about 100 years.
The second aspect of the present invention provides a kind of for genomic DNA long-term preservation solid phase adsorption solution, the solid phase
Adsorbent solution includes solid phase adsorption carrier, PEG8000 and NaCl.
In another preferred example, the solid phase adsorption carrier is nano silica microsphere.
In another preferred example, a diameter of 5~100nm of the silicon dioxide microsphere；Preferably 10~50nm；More preferably
About 20nm.
In another preferred example, the PEG is PEG8000.
In another preferred example, the solid phase adsorption pH value of solution ＜ 7.
In another preferred example, the solid phase adsorption pH value of solution is 4.5~6.5；Preferably 5.0~6.0；More preferably
5.5。
In another preferred example, the content of solid phase adsorption carrier is 0.05g/ml to 0.5g/ in the solid phase adsorption solution
ml；Preferably 0.1g/ml to 0.2g/ml.
In another preferred example, the content of PEG is 5%~40% (W/V) in the solid phase adsorption solution；Preferably 15%
~20% (W/V).
In another preferred example, the content of NaCl is 0.5~3M in the solid phase adsorption solution；Preferably 1~2M.
In another preferred example, the solid phase adsorption solution further includes Mg2+And Co2+, and Mg2+Molar concentration and Co2+It rubs
Your concentration ratio is 1~3:1；Preferably 3:2.
In another preferred example, Mg in the solid phase adsorption solution2+A concentration of 0.01~0.05mM；Preferably 0.03mM.
In another preferred example, Co in the solid phase adsorption solution2+A concentration of 0.005~0.03mM；Preferably 0.02mM.
In another preferred example, the solid phase adsorption solution includes：
Silicon dioxide microsphere 0.1g/ml, PEG80005%-40% (W/V) and NaCl 0.5-3M.
In another preferred example, further include in the solid phase adsorption solution：MgCl20.01~0.05mM and CoCl20.005
~0.03mM.
The third aspect of the present invention, provides a kind of long-term preservation kit of genomic DNA, and the kit includes this
Solid phase adsorption solution described in invention second aspect.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Description of the drawings
Fig. 1 is the electron microscopic picture of the nanometer silicon dioxide particle of the present invention.
Fig. 2 shows typical sample electrophoresis result.
Specific implementation mode
The present inventor's in-depth study by extensive by, obtains a kind of long-term preservation method of genomic DNA, experimental result
Show to preserve genome DNA using the solid phase material absorption of the present invention, the complete of genomic DNA structure can be kept for a long time
Property simultaneously keeps initial physicochemical property and its biological nature.
Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this
Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and
And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein all have with scientific terminology such as fields of the present invention
The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated
" about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes
101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).
Although can be used and heretofore described similar or of equal value any method in the implementation or test of the present invention
And material, herein place enumerate preferred method and material.
The present invention is using nano silica microsphere as carrier (nanoscale SiO2) gene is preserved, by testing repeatedly
The each component needed in solid phase adsorption solution is determined, and optimizes its content.When carrier adsorption gene and then carry out vacuum
The dehydration of concentration, the target gene preserved in this way will be firmly combined together with carrier.Acceleration study shows the present invention
Method can stablize preserve gene up to 35 years or more.
Specifically, the present inventor in the course of the research screens a variety of solid phase adsorption carriers, compared carbon nanotube,
A variety of DNA solid phase adsorptions carriers such as silica, iron oxide, calcium phosphate finally found that nano silica microsphere is solid as DNA
Phase absorption carrier has preferable Gene conservation effect, coordinates nano silica microsphere to solid phase adsorption solution on this basis
In other components carried out screening and optimizing, the solid phase adsorption solution obtained at present is estimated to be protected within 35 years or more time
Target gene stable in physicochemical property is held, and conventional atmospheric is kept in dark place.
Further verification test is found, is preserved target gene using the solid phase adsorption solution tentatively optimized, is protected for a long time
In the case of depositing, the genetic diversity decrease to some degree of target gene, in order to solve this problem, the present inventor is by anti-
Retrial, which is tested, finally to be had been surprisingly found that, a small amount of Mg is added in solid phase adsorption solution2+And Co2+It can make the heredity of target gene
Diversity keeps stable in a long time.On this basis, the present invention is completed.
Main advantages of the present invention are：
(1) target gene conventional atmospheric preserves in the present invention, thus significantly reduces Gene conservation cost.
(2) SiO that the present invention selects2Material physicochemical property and its stabilization, it is contemplated that effective holding time is up to century-old.It uses
The solid phase material absorption of the present invention preserves genome DNA, do not interfere with the integrality of genomic DNA structure, physicochemical property and
Its biological nature.
(3) gene in the prior art directly preserves, few due to measuring, and causes naked eyes invisible, later-stage utilization is formed
Certain obstacle；Target gene is stored on macroscopic solid phase adsorption carrier nano silica microsphere by the present invention, because
And greatly facilitate later stage use；
With reference to specific embodiment, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip
The works such as part such as U.S. Sambrook.J《Molecular Cloning: A Laboratory room guide》(Huang Peitang etc. is translated, Beijing：Science Press, 2002)
Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight
It calculates.Experiment material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.The present invention is implemented
The nano silica microsphere (grain size 20nm) used in example can be purchased from Hao Tian nanosecond science and technology (Shanghai) Co., Ltd..
Embodiment 1
Staphylococcus aureus (Staphylococcus aureus) is a kind of important pathogen of the mankind, is under the jurisdiction of Portugal
Grape Coccus (Staphylococcus) has the nickname of " thermophilic meat bacterium ", is the representative of gram-positive bacteria, can cause many serious
Infection.Therefore, it for the detection of staphylococcus aureus in environmental microorganism, is of great significance.Heat stable nuclease gene nuc
Site ensures that staphylococcus aureus has heat resistance, therefore, heat stable nuclease base in the chromosome of staphylococcus aureus
Because nuc can be as the key gene of staphylococcus aureus identification.In the present embodiment, solid phase material using the present invention
The staphylococcus aureus gene group total DNA preserved is adsorbed, with test cdna preservation effect.
(1) genome DNA is extracted
Staphylococcus aureus strains (ATCC 25923) culture solution of routine culture is diluted to 5 × 103A/mL, sample
Genome DNA extracts kit (the being purchased from Invitrogen) extraction of product total DNA.Sample is drawn to 2ml sterilizings with pipettor
In centrifuge tube, buffer solution is then added, is placed in 95 DEG C of water-baths 5 minutes, operation later is carried out by kit specification, extracts gene
Group total DNA.
(2) preservation and elution of target dna
By target dna, 100ul is settled to sterile ultra-pure water.100ul solid phase adsorption solution (0.1g/ml is added
SiO2, PEG800025%, NaCl 2M, pH 5.5), it mixes well, genomic DNA is allowed adequately to be connect with solid phase adsorption solution
It touches, the time is in 5min or more.Solid phase carrier is allowed adequately to adsorb genomic DNA.10000g centrifuges 5min, removes supernatant, about
200ul liquid all removes.200ul is added, 65%-90% alcohol washs solid phase material, and pipettor piping and druming is uniform.10000g
5min is centrifuged, supernatant is removed, liquid all removes, for the solid phase material after wash clean absorption genomic DNA.It is added
200ul, 65%-90% alcohol wash solid phase material, and pipettor piping and druming is uniform, convenient for transfer.
Solid phase suspension is all transferred in drying bottle, drying is concentrated in vacuo.Drain all liq ingredient, dry solid phase material
Material and genomic DNA are attached together.The solid phase material for being adsorbed with genomic DNA is preserved for use.
The elution process of target dna is as follows when use：
10000g first centrifuges 3min centrifugations and preserves pipe, it is ensured that all solids optical content is all in bottom.100ul is added
Ultra-pure water, fully shaking mixing can be blown and beaten with pipettor, be placed at room temperature for 5min.10000g centrifuges 10min, takes supernatant 80ul, i.e.,
DNA is obtained in supernatant.According to service condition, 80ul DNA can suitably be dispensed.
(3) PCR amplification
Pcr amplification reaction system is 50 μ the L, (Mg containing 10 × buffer solution2+20mmol/L) 5 μ L, dNTPs0.2mmol/L, draws
Object 1 μm of ol/L, Taq enzyme 5U, template about 50ng.Amplification condition：95 DEG C of pre-degenerations 3min, 94 DEG C of denaturation 40s, 52 DEG C of annealing 40s,
72 DEG C of extension 40s carry out 25 cycles, last 72 DEG C of extensions 7min.
Sense primer：5'-GCTTCTTTGCCAAATGGTTGTAC-3'(SEQ ID NO.:1)
Downstream primer：5'-TGTGGATGGTGATACATTTATTGCA-3'(SEQ ID NO.:2)
PCR amplification result is as shown in table 1 below, three repetitions of every group of sample.Sample 1 to sample 5 is the gene of routine preservation
Group total DNA.Sample 1 is the fresh genome DNA that step (1) obtains.Sample 2 to sample 5 is without using solid phase adsorption material
Material is directly concentrated in vacuo dry control group.Wherein, sample 2 is placed 3 months for -20 DEG C；Sample 3 is 4 DEG C and places 3 months；Sample
Product 4 are to be placed at room temperature for 3 months；Sample 5 is 60 DEG C and places 3 months.Sample 6 to sample 10 is the base that solid phase material absorption preserves
Because of a group total DNA, wherein sample 6 is to be placed at room temperature for 10 days；Sample 7 is 60 DEG C and places 10 days；Sample 8 is 60 DEG C and places 1 month；
Sample 9 is 60 DEG C and places 10 months；Sample 10 is 60 DEG C and places 30 months.
Table 1 preserves the PCR amplification result of DNA
| Sample number into spectrum | PCR amplification result | Sample number into spectrum | PCR amplification result |
| 1 | +++ | 6 | +++ |
| 2 | +++ | 7 | +++ |
| 3 | ―++ | 8 | +++ |
| 4 | ――+ | 9 | +++ |
| 5 | ――― | 10 | +++ |
Note：Each sample sets 3 groups of parallel, negative amplifications of "-" expression；"+" indicates positive amplification result.Fig. 2 is shown
For typical sample electrophoresis as a result, wherein swimming lane 1 is the electrophoresis result of sample 1, swimming lane 2 is the electrophoresis result of sample 2, swimming lane 3
For the positive electrophoresis result in sample 3, swimming lane 4 is the electrophoresis result of sample 6, and swimming lane 5 is the electrophoresis result of sample 7, and swimming lane 6 is
The electrophoresis result of sample 10.
PCR amplification the result shows that, using the present invention solid phase material absorption preserve genome DNA, in acceleration environment
It is placed under (60 DEG C) 30 months, still can carry out normal PCR amplification.Therefore, it is possible to predict, at room temperature, place 35 years
Genome sample still can be normally carried out PCR amplification.And the genome of the drying regime without solid phase material absorption preservation is total
DNA is only capable of the time of storage some months at room temperature.
(4) plasmid construction
After the genome DNA elution that solid phase material absorption is preserved, both ends add BamHI restriction enzyme sites, are cloned into matter
Grain carrier pUC19 (is purchased from TAKARA companies), and structure obtains the plasmid for including target gene sequence.Culture contains gained plasmid
Recombination bacillus coli extracts plasmid using plasmid extraction kit (OMEGA).
The experimental results showed that even if under high temperature acceleration environment (60 DEG C are placed 30 months), the solid phase material of the present invention is used
The genome DNA that material absorption preserves can also be normally carried out clone, and the plasmid of extraction, which is sent to Shanghai Mei Ji biotechnologys, to be had
Limit company is sequenced, and sequencing result shows that sequence 100% is correct.Therefore, base is preserved using the solid phase material absorption of the present invention
Because of a group total DNA, do not have an impact the integrality, physicochemical property and its biological nature of genomic DNA structure.
Embodiment 2
The type and quantity of edaphon change with the difference of soil-forming conditions and its soil depth, generally with bacterial population
Amount is most.The genetic diversity of edaphon is all kinds of inhereditary materials and heredity that edaphon carries at the genetic level
The summation of information is being finally reflected for microbial diversity.In the present embodiment, what solid phase material absorption using the present invention preserved
Microbial genome total DNA in soil sample.
(1) sample total DNA acquires
The pedotheque of 5 centimeters or so depth of earth's surface, is placed in the sample box of sterilizing at the shady and cool humidity of acquisition.
Pedotheque total DNA is extracted with kit (Qiagen QIAamp DNA Stool Minikits), and operation is normally
Bright book carries out.The absorption of target dna is preserved to be carried out with reference to step (2) in embodiment 1.
(2) sample Diversity Detection
Use universal primer PCR amplification bacterial 16 S rDNA:(5'-6-FAM-AGAGTTTGATCCTGGCTCAG-3'(SEQ
ID NO.:And 5'-ACGGTTACCTTGTTACGACTT-3'(SEQ ID NO. 3):4)).
Pcr amplification product is returned through 1% agarose gel electrophoresis, with gel reclaims kit (TIANGEN) from agarose
Receive target DNA fragment.The PCR recovery products marked to fluorescent primer using restriction enzyme (BioLabs) carry out enzyme respectively
It cuts.85 DEG C of inactivation 15min of digestion products.It is added 95% ethyl alcohol of 2 μ l 3mol/L sodium acetates (pH 5.2) and 50 μ l precoolings, 4 DEG C
Lower 16000g centrifuges 30min, removes supernatant.Then it is washed twice with 70% ethyl alcohol of 200 μ l precoolings, by DNA sample weight after air-drying
It is dissolved in 15 μ l distilled waters.The PCR product of 50ng or so is taken to carry out Capillary Electrophoresis on ABI 3730xl automatic sequencers.Point
Obtained T-RFLP collection of illustrative plates after analysis Capillary Electrophoresis, sets 100 flat fluorescents as baseline, T-RFs peak heights>100 flat fluorescents
T-RFs regards as effective T-RFs, the wherein quantity of abundance, that is, T-RFs, is referred to using the abundance of each sample as genetic diversity
Number.It is for statistical analysis with SPSS softwares.
Sample 1-1 is the fresh total DNA of soil sample obtained.Sample 1-2 to sample 1-5 is without using solid phase adsorption material
Material is directly concentrated in vacuo dry control group total DNA.Wherein, sample 1-2 is placed 3 months for -20 DEG C；Sample 1-3 is 4 DEG C and puts
It sets 3 months；Sample 1-4 is to be placed at room temperature for 3 months；Sample 1-5 is 60 DEG C and places 3 months.Sample 2-1 to sample 2-5 is solid
Soil sample total DNA (the solid phase adsorption solution SiO containing 0.1g/ml used that phase material absorption preserves2, PEG800025%,
NaCl 2M, pH 5.5), sample 2-1 is to be placed at room temperature for 10 days；Sample 2-2 is 60 DEG C and places 10 days；Sample 2-3 is 60 DEG C of placements
1 month；Sample 2-4 is 60 DEG C and places 10 months；Sample 2-5 is 60 DEG C and places 30 months.Sample 3-1 to sample 3-5 is solid
Soil sample total DNA (the solid phase adsorption solution SiO containing 0.1g/ml used that phase material absorption preserves2, PEG800025%,
NaCl 2M, pH 5.5, and add MgCl20.03mM、CoCl20.02mM), sample 3-1 is to be placed at room temperature for 10 days；Sample 3-2 is
60 DEG C are placed 10 days；Sample 3-3 is 60 DEG C and places 1 month；Sample 3-4 is 60 DEG C and places 10 months；Sample 3-5 is 60 DEG C of placements
30 months.
Testing result is as shown in table 2.
2 sample DNA Diversity Detection of table
| Sample number into spectrum | Abundance | Sample number into spectrum | Abundance | Sample number into spectrum | Abundance |
| 1-1 | 38 | 2-1 | 38 | 3-1 | 38 |
| 1-2 | 36 | 2-2 | 36 | 3-2 | 37 |
| 1-3 | 13 | 2-3 | 32 | 3-3 | 37 |
| 1-4 | 8 | 2-4 | 25 | 3-4 | 37 |
| 1-5 | 0 | 2-5 | 18 | 3-5 | 36 |
Testing result shows that the Initial abundance of microorganism in acquired soil sample is 38, is preserved in control group total DNA
Under the conditions of, as the extension of holding time or temperature increase, the bacterial abundance detected significantly reduces, and can not react true
Soil sample in microorganism Initial abundance.The testing result of sample 2-1 to 2-5 is shown to adsorb using solid phase material and is protected
The soil sample genome DNA deposited can greatly extend the holding time of sample DNA, significantly delay genetic diversity
It reduces.But after being placed 30 months under acceleration environment (60 DEG C), sample abundance reduces 52%, and sample genomic information is lost
It loses still more.In order to continue to improve Gene conservation effect, inventor improves solid phase adsorption solution, is had been surprisingly found that
Solid phase adsorption solution adds other metal ions and is significantly affected to Gene conservation effect, and then is sent out by lot of experiment validation
A small amount of MgCl is added in now original solid phase adsorption solution simultaneously2And CoCl2There is actively impact to Gene conservation, pass through
It advanced optimizes and MgCl is determined2Additive amount is 0.01~0.05mM and CoCl2Additive amount is 0.005~0.03mM.Sample 3-1
Testing result to 3-5 has been reacted using the solid phase adsorption solution preservation sample DNA effect after optimization, the results showed that, even if
After being placed 30 months under acceleration environment (60 DEG C), bacterial abundance reduced by only about 5%, can react micro- in initial sample substantially
Biological abundance.Therefore, the sample genome DNA preserved using the optimized solid phase material absorption of the present invention, can be true
Also original initial DNA information so that initial DNA information will not be cut down with the extension of time.
Embodiment 3
Human oral cavity mucomembranous epithelial cell genomic DNA preserves
It is scraped for several times in oral cavity wall with sterile nylon brush, taking-up is immersed in 500 μ l cell pyrolysis liquids, mild agitation；
Be added Proteinase K Solution (20mg/ml, the green skies) 1.5 μ l simultaneously, after mixing in 55 DEG C of water-baths incubated overnight so that cell
Fully cracking, disengages genomic DNA.
Ribonuclease A solution (4mg/ml, Thermo) 1.5 μ l are added in cell lysates, 37 DEG C of water-baths after mixing
Heat preservation, so that RNA degrades.
Protein precipitant (ammonium acetate) 100 μ l are added in above-mentioned reaction solution, fully shakes up, makes protein precipitation, from
The protein of heart removal precipitation, stays the supernatant containing DNA.It is settled to 100ul with sterile ultra-pure water.100ul solid phases are added to inhale
Attached solution (0.1g/ml SiO2, PEG800025%, NaCl 2M, MgCl20.03mM, CoCl20.02mM, pH 5.5), it is fully mixed
It is even, allow genomic DNA adequately and the contact of solid phase adsorption solution, the time is in 5min or more.10000g centrifuge 5min, remove on
Clearly, about 200ul liquid all removes.200ul is added, 65%-90% alcohol washs solid phase material, and pipettor piping and druming is uniform.
10000g centrifuges 5min, removes supernatant, and liquid all removes, for the solid phase material after wash clean absorption genomic DNA.Add
Enter 200ul, 65%-90% alcohol washs solid phase material, and pipettor piping and druming is uniform.Solid phase suspension is all transferred in drying bottle,
It is concentrated in vacuo drying.All liq ingredient is drained, dry solid phase material and genomic DNA are attached together.Base will be adsorbed with
Because the solid phase material room temperature of group DNA preserves for use.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Sequence table
<110>It is safe and comfortable（Shanghai）Bio tech ltd
<120>The store method and kit of long-term gene group DNA
<130> 170233ZI
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>Staphylococcus aureus (Staphylococcus aureus)
<400> 1
gcttctttgc caaatggttg tac 23
<210> 2
<211> 25
<212> DNA
<213>Staphylococcus aureus (Staphylococcus aureus)
<400> 2
tgtggatggt gatacattta ttgca 25
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
agagtttgat cctggctcag 20
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
acggttacct tgttacgact t 21
Claims (10)
1. a kind of long-term preservation method of genomic DNA, which is characterized in that the method includes the steps：
(1) solid phase carrier adsorbs
Genomic DNA to be saved is provided, is mixed being mixed well in genomic DNA addition solid phase adsorption solution
Liquid；Wherein, the solid phase adsorption solution includes solid phase adsorption carrier, PEG and NaCl；
(2) it washs
The mixed liquor obtained in centrifugation step (1) removes supernatant；Organic solvent washing precipitation is added, supernatant is removed after centrifugation
Organic solvent mixing is added in liquid；
(3) dry
The liquid that step (2) obtains is transferred to and is preserved in pipe, dry removal liquid component, to realize to genomic DNA
Long-term preservation.
2. the method as described in claim 1, which is characterized in that the solid phase adsorption carrier is nano silica microsphere.
3. method as claimed in claim 2, which is characterized in that a diameter of 5~100nm of nano silica microsphere；It is excellent
It is selected as 10~50nm.
4. the method as described in claim 1, which is characterized in that the PEG is PEG8000.
5. the method as described in claim 1, which is characterized in that the solid phase adsorption pH value of solution ＜ 7.
6. the method as described in claim 1, which is characterized in that the content of solid phase adsorption carrier is in the solid phase adsorption solution
0.05g/ml to 0.5g/ml (preferably 0.1g/ml to 0.2g/ml)；In the solid phase adsorption solution content of PEG be 5%~
40% (W/V)；And/or the content of NaCl is 0.5~3M in the solid phase adsorption solution；Preferably 1~2M.
7. the method as described in claim 1, which is characterized in that the solid phase adsorption solution further includes Mg2+And Co2+, and Mg2 +Molar concentration and Co2+Molar concentration rate is 1~3:1.
8. one kind being used for genomic DNA long-term preservation solid phase adsorption solution, which is characterized in that the solid phase adsorption solution includes solid
Phase absorption carrier, PEG8000 and NaCl.
9. solid phase adsorption solution as claimed in claim 8, which is characterized in that the solid phase adsorption carrier is nano silicon dioxide
Microballoon.
10. a kind of long-term preservation kit of genomic DNA, which is characterized in that the kit includes according to any one of claims 8
Solid phase adsorption solution.
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| CN113249433A (en) * | 2021-05-31 | 2021-08-13 | 安康（上海）生物科技有限公司 | Gene long-term storage method and kit |
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