CN108866047A - A kind of genome DNA extracting method based on multigelation mode - Google Patents
A kind of genome DNA extracting method based on multigelation mode Download PDFInfo
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Abstract
The invention discloses a kind of genome DNA extracting methods based on multigelation mode, to extraction cell culture (bacterium, fungi, nematode etc.) Extraction buffer is added, twice, 37 DEG C of incubation 10min of RNase are added in multigelation after mixing, it adds after 5M NaCl and 10%SDS mix well, the mixed solution of phenol chloroform-isoamyl alcohol (25: 24: 1), extracting is added in freeze thawing again；Chloroform-isoamyl alcohol (24: 1) mixed solution, extracting is added；Cold ethyl alcohol and sodium acetate is added, is centrifuged, precipitating drying at room temperature after ethanol washing；Add ddH2O or TE buffer solution DNA.This method is based on multigelation mode and carries out, widely applicable, and DNA mass that is high-efficient, extracting is high, has good versatility.
Description
Technical field
The invention belongs to the extracting methods of DNA, and in particular to a kind of extracting genome DNA side based on multigelation mode
Method.
Technical background
With the development of molecular biology technology, the successful extraction of DNA has become the basis and pass of related science research
Key.It extracts genomic DNA and is usually applied to building genomic library, genome sequencing, Southern hybridization, RFLP and PCR
Amplification etc..The DNA of rapidly extracting high quality genome will be the research of species affiliation, genome comparison, genetic diversity
Reference is provided with the subsequent molecular biology research such as the label of certain specific genes.Especially in carrying out population genetic study
The research sample size needed is big, in addition to the quality to DNA and other than quantity requires, it is also necessary to which method is simple.
Conventional method on general molecular biology experiment guiding book, due to variety classes or same kind of different tissues
Because of its eucaryotic cell structure and the difference of ingredient, need to carry out DNA extraction using different methods to different species respectively, such as
Extracting for bacterial genomes DNA need to mostly use greatly polishing etc. using the extraction of CTAB method, nematode gene group DNA；It is existing on the market
Some DNA extraction kits, also tend to just for single species.As for gram-positive bacterium or gramnegative bacterium
DNA extraction kit etc. is rarely reported the extraction that using a kind of method different plant species are carried out with DNA.
The genome DNA extracting method complex steps of routine, time-consuming.Generally require time-consuming 3~5 hours.And DNA is pure
Spend it is not high, it is more or less during DNA is extracted all to encounter the pollution of the substances such as protein, carbohydrate, phenols and RNA.
Especially carbohydrate and phenolic substances influence have very strong inhibiting effect to endonuclease, generate to Southern hybridization larger
Influence；It also have a large impact on common PCR amplification, and the presence of glucide will affect the unwinding and Taq enzyme of DNA
Activity.When encountering that sugar content is higher or the higher species of phenols content, conventional method is difficult to be able to satisfy full-length genome survey
The requirement of sequence high-purity, large fragment.So how it is simple, fast and efficient remove these substances pollution, to purification genome
It is most important for DNA.
Summary of the invention
Goal of the invention：In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide one kind based on repeatedly
The genome DNA extracting method of freeze thawing mode, the DNA that can break between different plant species are extracted, and step is simple, extraction process is time-consuming
Short, the content that can get high-purity genomic DNA (being practically free of the substances such as protein, carbohydrate, phenols and RNA) and DNA is full
The general molecular biology experiment operation such as sufficient genome sequencing library construction, Southern hybridization, RFLP and regular-PCR amplification
Requirement.
Technical solution：In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is：
A kind of genome DNA extracting method based on multigelation mode, steps are as follows：
(1) histocyte of DNA to be extracted is collected in centrifuge tube, and Extraction buffer is added, mixes well, multigelation
2 times；
(2) RNase is added in the mixed solution obtained to step (1) to mix well, is incubated for；
(3) after addition NaCl and SDS is mixed well in the mixed solution obtained to step (2), freeze thawing again；
(4) mixed solution of isometric phenol chloroform-isoamyl alcohol is added to the mixed solution that step (3) obtains, extracts,
Centrifuging and taking supernatant；
(5) isometric chloroform-isoamyl alcohol is added in the supernatant solution obtained to step (4), extracts, centrifuging and taking supernatant；
(6) cold ethyl alcohol and sodium acetate aqueous solution is added in the mixed solution obtained to step (5), is centrifuged, precipitating；
(7) in the precipitating obtained to step (6) be added ethanol wash after drying at room temperature, add ddH2O or TE buffer solution
DNA。
In step (1), the Extraction buffer configures obtain as follows：By NaCl 4g, CTAB 10g, poly- second
Alkene pyrrolidone 2g, the Tris-HCl buffer 10mL of 1mol/L pH8.0,1mol/L pH8.0 EDTA 2mL be dissolved in ddH2O
In, constant volume to 100mL.
In step (1), histocyte includes bacterium and fungi.
In step (1), 400 μ L of Extraction buffer is added in every 4~6mL histocyte bacterium solution.
In step (2), every 4~6mL histocyte bacterium solution is added 5 μ L 10mg/mL RNase and mixes well, 37 DEG C of incubations
10min。
In step (3), 50 μ L 5M NaCl and 100 μ L 10%SDS are added in every 4~6mL histocyte bacterium solution.
In step (4), phenol-chlorine that isometric volume ratio is 25: 24: 1 is added in the mixed solution obtained to step (3)
The mixed solution of imitative-isoamyl alcohol, extracting, 12000rpm are centrifuged 10min, take supernatant.
In step (5), it is different that the chloroform-that isometric volume ratio is 24: 1 is added in the supernatant solution that obtains to step (4)
Amylalcohol, extracting, 12000rpm are centrifuged 10min, take supernatant；It is repeated twice, wherein second of centrifugation 5min.
In step (6), the cold ethyl alcohol and 0.1 times of volume of 2 times of volumes is added in the mixed solution that obtains to step (5)
3mol/L sodium acetate aqueous solution is centrifuged, precipitating.
Drying at room temperature after 70% ethanol washing is added in step (7), in the precipitating that obtains to step (6), adds ddH2O or
TE buffer solution DNA.
Beneficial effect：Compared with prior art, the genome DNA extracting method of the invention based on multigelation mode,
It has the advantage that：
1) the method for the present invention is suitable for the gene of a variety of species such as bacterium (Gram-positive, negative bacteria), fungi, nematode
Group DNA is extracted, and has certain popularity.
2) genomic DNA extracted using the method for the present invention is high compared to traditional CTAB method, RNA isolation kit concentration, favorably
In the subsequent molecular biology experiment of progress.
3) using the method for the present invention extract genomic DNA purity it is higher, integrality is preferable, meet Southern Blot,
The demand of genome sequencing.
4) the method for the present invention is equally applicable for extracting the genomic DNA of the high species of sugar content.
5) shorter using the time used in the method for the present invention extraction genomic DNA, save the time.
6) not the problem of the method for the present invention does not add the biological reagents such as lysozyme, Proteinase K, avoids DNA cryo-conservation, and
Certain cost is saved for relevant molecular biology experiment.
7) step of the present invention is simple, easy to operate, it can be achieved that high quality extracting genome DNA.
Detailed description of the invention
Fig. 1 is 1% agarose gel electrophoresis figure for the DNA that embodiment 1 is extracted；
Fig. 2 is 1% agarose gel electrophoresis figure for the DNA that embodiment 2 is extracted；
Fig. 3 is 1% agarose gel electrophoresis figure for the DNA that embodiment 3 is extracted；
Fig. 4 is 1% agarose gel electrophoresis figure for the DNA that embodiment 4 is extracted；
Fig. 5 is 1% agarose gel electrophoresis figure after 5 DNA cloning of embodiment；
Specific embodiment
The present invention is described further combined with specific embodiments below.In following embodiment, used extract is delayed
Fliud flushing, configuration obtains as follows：By NaCl 4g, CTAB 10g, polyvinylpyrrolidone 2g, 1mol/L pH8.0
Tris-HCl buffer 10mL, 1mol/L pH8.0 EDTA 2mL be dissolved in ddH2In O, constant volume to 100mL.
1 Gram-positive of embodiment, the extracting genome DNA of negative bacteria (see Fig. 1)
1, gramnegative bacterium (Burkholderia pyrrocinia JK-SH007, Burkholderia pyrrocinia
JK-SH007) extracting genome DNA, steps are as follows：
(1) 3~6mL Burkholderia pyrrocinia JK-SH007 is incubated overnight, and 10000rpm is centrifuged 2min, abandons supernatant；
(2) 400 μ L of Extraction buffer is added in the cell obtained to step (1), mixes well, 2 (this realities of multigelation
The jelly for applying example is liquid nitrogen flash freezer, melts and melts for room temperature, and refrigerator freezing can also be used and melt in room temperature to 60 DEG C or less)；
(3) mixed solution obtained to step (2) is added 5 μ L 10mg/mL RNase and mixes well, 37 DEG C of incubations
10min；
(4) after the mixed solution addition 50 μ L 5M NaCl and 100 μ L 10%SDS obtained to step (3) are mixed well,
Freeze thawing again；
(5) mixing that isometric phenol chloroform-isoamyl alcohol (25: 24: 1) is added in the mixed solution obtained to step (4) is molten
Liquid extracts, and centrifugation takes supernatant；It is repeated twice；
(6) it is added isometric chloroform-isoamyl alcohol (24: 1), extracts in the supernatant solution obtained to step (5), centrifugation,
Take supernatant；
(7) the cold ethyl alcohol of 2 times of volumes and the sodium acetate of 0.1 times of volume is added in the mixed solution obtained to step (6)
(3mol/L) aqueous solution is centrifuged, precipitating；
(8) in the precipitating obtained to step (7) be added 70% ethanol washing after drying at room temperature, add ddH2O or TE buffering
Liquid dissolving DNA.
2, gram-positive bacterium (bacillus pumilus Bacillus pumilus HRl0) extracting genome DNA, step
It is as follows：
(1) 3~6mL bacillus pumilus Bacillus pumilus HR10 is incubated overnight, and 10000rpm is centrifuged 2min,
Abandon supernatant；
(2) 400 μ L of Extraction buffer is added in the cell obtained to step (1), mixes well, multigelation 2 times；
(3) mixed solution obtained to step (2) is added 5 μ L 10mg/mL RNase and mixes well, 37 DEG C of incubations
10min；
(4) after the mixed solution addition 50 μ L 5M NaCl and 100 μ L 10%SDS obtained to step (3) are mixed well,
Freeze thawing again；
(5) mixing that isometric phenol chloroform-isoamyl alcohol (25: 24: 1) is added in the mixed solution obtained to step (4) is molten
Liquid extracts, and centrifugation takes supernatant；It is repeated twice；
(6) it is added isometric chloroform-isoamyl alcohol (24: 1), extracts in the supernatant solution obtained to step (5), centrifugation,
Take supernatant；
(7) the cold ethyl alcohol of 2 times of volumes and the sodium acetate of 0.1 times of volume is added in the mixed solution obtained to step (6)
(3mol/L) aqueous solution is centrifuged, precipitating；
(8) in the precipitating obtained to step (7) be added 70% ethanol washing after drying at room temperature, add ddH2O or TE buffering
Liquid dissolving DNA.
As a result as shown in Figure 1, in figure, swimming lane 1：Burkholderia pyrrocinia JK-SH007 extracting genome DNA result；
Swimming lane 2：Bacillus pumilus Bacillus pumilus HR10 extracting genome DNA illustrates this as a result, band is single bright
Invention has certain popularity, can meet the genomic DNA of gramnegative bacterium and gram-positive bacterium simultaneously
It extracts.
Embodiment 2
Bacterium (Burkholderia pyrrocinia JK-SH007), fungi (Botrytis cinerea Botrytis cinerea) and nematode
The extracting genome DNA of (Bursaphelenchus xylophilus Bursaphelenchuh xylophilus), steps are as follows：
(1) it is raw that Burkholderia pyrrocinia JK-SH007 thallus, 1-2 culture dish that 3~6mL is incubated overnight are collected respectively
Long vigorous Botrytis cinerea (Botrytis cinerea), about 10000 Bursaphelenchus xylophiluses are in 1.5mL centrifuge tube；
(2) 400 μ L of Extraction buffer is added in the cell obtained to step (1), mixes well, multigelation 2 times；
(3) mixed solution obtained to step (2) is added 5 μ L 10mg/mL RNase and mixes well, 37 DEG C of incubations
10min；
(4) after the mixed solution addition 50 μ L 5M NaCl and 100 μ L 10%SDS obtained to step (3) are mixed well,
Freeze thawing again；
(5) mixing that isometric phenol chloroform-isoamyl alcohol (25: 24: 1) is added in the mixed solution obtained to step (4) is molten
Liquid extracts, and centrifugation takes supernatant；It is repeated twice；
(6) it is added isometric chloroform-isoamyl alcohol (24: 1), extracts in the supernatant solution obtained to step (5), centrifugation,
Take supernatant；
(7) the cold ethyl alcohol of 2 times of volumes and the sodium acetate of 0.1 times of volume is added in the mixed solution obtained to step (6)
(3mol/L) aqueous solution is centrifuged, precipitating；
(8) in the precipitating obtained to step (7) be added 70% ethanol washing after drying at room temperature, add ddH2O or TE buffering
Liquid dissolving DNA；
(9) concentration and electrophoresis detection are carried out to the DNA of said extracted.
1 DNA extracting concentration of table, purity
Remarks：1：Bursaphelenchus xylophilus；2：Botrytis cinerea；3：Burkholderia pyrrocinia JK-SH007
As a result as shown in Figure 2 and Table 1, in figure, swimming lane 1：Bursaphelenchus xylophilus extracting genome DNA is as a result, swimming lane 2：Grey grape
Spore extracting genome DNA is as a result, swimming lane 3：Burkholderia pyrrocinia JK-SH007 extracting genome DNA is as a result, the present invention
The extraction that the genomic DNA of bacterium, fungi and nematode can be met simultaneously illustrates that the present invention has certain popularity.
Embodiment 3
The high bacterial genomes DNA of sugar content is extracted with CTAB+SDS method using the method for embodiment 1 to be compared.
1, histiocytic genomic DNA is extracted using the present processes, steps are as follows：
(1) 3~6mL histocyte is incubated overnight, and 10000rpm is centrifuged 2min, abandons supernatant；
(2) 400 μ L of Extraction buffer is added in the cell obtained to step (1), mixes well, multigelation 2 times；
(3) mixed solution obtained to step (2) is added 5 μ L 10mg/mL RNase and mixes well, 37 DEG C of incubations
10min；
(4) after the mixed solution addition 50 μ L 5M NaCl and 100 μ L 10%SDS obtained to step (3) are mixed well,
Freeze thawing again；
(5) mixing that isometric phenol chloroform-isoamyl alcohol (25: 24: 1) is added in the mixed solution obtained to step (4) is molten
Liquid extracts, and centrifugation takes supernatant；(being repeated twice)
(6) it is added isometric chloroform-isoamyl alcohol (24: 1), extracts in the supernatant solution obtained to step (5), centrifugation,
Take supernatant；
(7) the cold ethyl alcohol of 2 times of volumes and the sodium acetate of 0.1 times of volume is added in the mixed solution obtained to step (6)
(3mol/L) aqueous solution is centrifuged, precipitating；
(8) in the precipitating obtained to step (7) be added 70% ethanol washing after drying at room temperature, add ddH2O or TE buffering
Liquid dissolving DNA.
2, identical histiocytic genomic DNA is extracted using CTAB+SDS method, method is the same as [stalks of rice, wheat, etc. is outstanding, and Li Li, Tang Qiong wait
Comparison [J] Journal of Natural Science of Hunan Normal University of bacterial genomes DNA extraction method, 2015,38 (6) are sequenced in the third generation：
14-20.]。
By taking Burkholderia pyrrocinia JK-SH007 as an example, as a result as shown in figure 3, in figure, swimming lane 1：Use the present invention
Extraction result of the method to Burkholderia pyrrocinia JK-SH007 genomic DNA；Swimming lane 2：Using CTAB-SDS method to pyrrole
The extraction of bulkholderia cepasea JK-SH007 genomic DNA is coughed up as a result, the genomic DNA for illustrating that the method for the present invention is extracted is compared
In traditional CTAB method purity is high, the pollution of the substances such as no protein, carbohydrate fully meets Southern blot, full-length genome
The requirement of sequencing.Be conducive to carry out subsequent molecular biology experiment；The method of the present invention is suitable for extracting the high species of sugar content
Genomic DNA.
2 present invention of table is compared with the genomic DNA concentration that traditional CT AB method is extracted
| 1DNA concentration | 21DNA concentration | 31DNA concentration | 41DNA concentration | |
| CTAB method | 349.08ng/μL | 433.93ng/μL | 221.90ng/μL | 279.81ng/μL |
| The method of the present invention | 1085.30ng/μL | 808.37ng/μL | 709.43ng/μL | 687.39ng/μL |
Remarks：1：Burkholderia pyrrocinia JK-SH007；2：Bacillus pumilus Bacillus pumilus
HRl0；3：Botrytis cinerea；4：Bursaphelenchus xylophilus.
With Burkholderia pyrrocinia JK-SH007, bacillus pumilus Bacillus pumilus HRl0, grey grape
It for spore and Bursaphelenchus xylophilus, is compared, the results are shown in Table 2, illustrates the genomic DNA of the method for the present invention extraction compared to biography
CTAB method, the polishing concentration of system are higher, are conducive to carry out subsequent molecular biology experiment
Compared with 3 the method for the present invention of table extracts the operating time of genomic DNA with traditional CT AB method
| Method | The method of the present invention | CTAB+SDS method | SDS method[1] | RNA isolation kit[1] |
| Time | 1.5-2h | 4.5-5h | 4-5h | 2-3h |
Note：CTAB+SDS method, SDS method, RNA isolation kit, with [stalks of rice, wheat, etc. is outstanding, Li Li, Tang Qiong, waits the third generation that bacterium base is sequenced
Because of comparison [J] Journal of Natural Science of Hunan Normal University of group DNA extraction method, 2015,38 (6)：14-20.].
By taking Burkholderia pyrrocinia JK-SH007 as an example, it is compared the time, the results are shown in Table 3, illustrates this hair
It is bright premised on the quality for guaranteeing DNA in the case where, step is easy, more saves the time.
Embodiment 4
By taking Botrytis cinerea Botrytis cinerea as an example, the present processes and liquid nitrogen grinding+CTAB extraction process are extracted
Genomic DNA integrality compare.
1, Botrytis cinerea Botrytis cinerea genomic DNA is extracted using the present processes, steps are as follows：
(1) the eugonic Botrytis cinerea of 1-2 culture dish (Botrytis cinerea) is collected in 1.5mL centrifuge tube；
(2) 400 μ L of Extraction buffer is added in the cell obtained to step (1), mixes well, multigelation 2 times；
(3) mixed solution obtained to step (2) is added 5 μ L 10mg/mL RNase and mixes well, 37 DEG C of incubations
10min；
(4) after the mixed solution addition 50 μ L 5M NaCl and 100 μ L 10%SDS obtained to step (3) are mixed well,
Freeze thawing again；
(5) mixing that isometric phenol chloroform-isoamyl alcohol (25: 24: 1) is added in the mixed solution obtained to step (4) is molten
Liquid extracts, and centrifugation takes supernatant；(being repeated twice)
(6) it is added isometric chloroform-isoamyl alcohol (24: 1), extracts in the supernatant solution obtained to step (5), centrifugation,
Take supernatant；
(7) the cold ethyl alcohol of 2 times of volumes and the sodium acetate of 0.1 times of volume is added in the mixed solution obtained to step (6)
(3mol/L) aqueous solution is centrifuged, precipitating；
(8) in the precipitating obtained to step (7) be added 70% ethanol washing after drying at room temperature, add ddH2O or TE buffering
Liquid dissolving DNA.
2, Botrytis cinerea Botrytis cinerea genomic DNA is extracted using liquid nitrogen grinding+CTAB extraction process, it is specific to walk
It is rapid with [research of microorganism total DNA extracting method and its apply the Changsha [D] Hunan University in Song Lin tinkling of pieces of jade agricultural waste material compost,
2008.]。
As a result as shown in figure 4, in figure, swimming lane 1：Using the method for the present invention to the extraction result of Botrytis cinerea genomic DNA；
Swimming lane 2：Using liquid nitrogen grinding+CTAB extraction process to the extraction of Botrytis cinerea genomic DNA as a result, the base that the method for the present invention is extracted
Because of a group DNA, integrality is preferable, meets the needs of Southern Blot, genome sequencing.
Embodiment 5
The Botrytis cinerea Botrytis cinerea and Bursaphelenchus xylophilus Bursaphelenchuh that embodiment 2 is extracted
Xylophilus genomic DNA carries out the PCR amplification of ITS-rDNA and the universal primer of product detection selection amplification ITS sequence
(ITS4/ITS7) 2 kinds of genomic DNAs are expanded.Amplification system is 2 × EasyTaq PCR SuperMix, 10 μ L, 10 μM
Each 1 μ L of primer, 1 μ L of template, ddH2O supplies 20 μ L；PCR reaction condition：94 DEG C of denaturation 5min are again with 94 DEG C of denaturation 30s, and 55 DEG C
Anneal 30s, 72 DEG C of extension 1.0min, 33 circulations, 72 DEG C of extension 10min.PCR product electrophoresis in 1% Ago-Gel,
It takes pictures under Gelred dyeing ultraviolet lamp.Successful amplification has gone out the ITS of Botrytis cinerea and Bursaphelenchus xylophilus to electrophoresis result as shown in Figure 5
Purpose band be respectively swimming lane 1 and 2.Stripe size is about 500bp-600bp or so, and band is single, bright.Illustrate using real
Applying the DNA extracted in example 2 can satisfy PCR sample requirement.
The PCR of 16s rDNA is carried out to the Burkholderia pyrrocinia JK-SH007 genomic DNA extracted in embodiment 2
Amplification and product detection.The universal primer (27F/1492R) of selection amplification bacterial 16 S rDNA expands genomic DNA.
Amplification system is 20 μ L systems：2 × EasyTaq PCR SuperMix10 μ L, each 1 μ L of 10 μM of primers, 1 μ L of template, ddH2O
Supply 20 μ L.PCR reaction condition：94 DEG C of denaturation 5min again with 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1.5min,
33 circulations, 72 DEG C of extension 10min.PCR product electrophoresis in 1% Ago-Gel, Gelred is dyed to take pictures under ultraviolet lamp.Electricity
Swim result such as Fig. 5, and Successful amplification goes out the purpose band 1500bp of the 16S rDNA of bacterium shown in swimming lane 3.Illustrate using real
Applying the DNA extracted in example 2 can satisfy PCR sample requirement.
Claims (10)
1. a kind of genome DNA extracting method based on multigelation mode, which is characterized in that steps are as follows：
(1) histocyte of DNA to be extracted is collected in centrifuge tube, and Extraction buffer is added, mixes well, multigelation 2 times；
(2) RNase is added in the mixed solution obtained to step (1) to mix well, is incubated for；
(3) after addition NaCl and SDS is mixed well in the mixed solution obtained to step (2), freeze thawing again；
(4) mixed solution of isometric phenol chloroform-isoamyl alcohol is added to the mixed solution that step (3) obtains, extracts, centrifugation
Take supernatant；
(5) isometric chloroform-isoamyl alcohol is added in the supernatant solution obtained to step (4), extracts, centrifuging and taking supernatant；
(6) cold ethyl alcohol and sodium acetate aqueous solution is added in the mixed solution obtained to step (5), is centrifuged, precipitating；
(7) in the precipitating obtained to step (6) be added ethanol wash after drying at room temperature, add ddH2O or TE buffer solution DNA.
2. the genome DNA extracting method according to claim 1 based on multigelation mode, which is characterized in that step
(1) in, the Extraction buffer configures obtain as follows：By NaCl 4g, CTAB 10g, polyvinylpyrrolidone 2g,
The Tris-HCl buffer 10mL of 1mol/L pH8.0, the EDTA 2mL of 1mol/L pH8.0 are dissolved in ddH2In O, constant volume is arrived
100mL。
3. the genome DNA extracting method according to claim 1 based on multigelation mode, which is characterized in that step
(1) in, histocyte includes bacterium, fungi and nematode.
4. the genome DNA extracting method according to claim 1 based on multigelation mode, which is characterized in that step
(1) in, 400 μ L of Extraction buffer is added in every 4~6mL histocyte bacterium solution.
5. the genome DNA extracting method according to claim 1 based on multigelation mode, which is characterized in that step
(2) in, every 4~6mL histocyte bacterium solution is added 5 μ L 10mg/mL RNase and mixes well, 37 DEG C of incubation 10min.
6. the genome DNA extracting method according to claim 1 based on multigelation mode, which is characterized in that step
(3) in, 50 μ L5MNaCl and 100 μ L10%SDS are added in every 4~6mL histocyte bacterium solution.
7. the genome DNA extracting method according to claim 1 based on multigelation mode, which is characterized in that step
(4) in, phenol chloroform-isoamyl alcohol that isometric volume ratio is 25: 24: 1 is added in the mixed solution obtained to step (3)
Mixed solution, extracting, 12000rpm are centrifuged 10min, take supernatant.
8. the genome DNA extracting method according to claim 1 based on multigelation mode, which is characterized in that step
(5) in, it is 24 that isometric volume ratio is added in the supernatant solution that obtains to step (4)：1 chloroform-isoamyl alcohol, extracting,
12000rpm is centrifuged 10min, takes supernatant；It is repeated twice, wherein second of centrifugation 5min.
9. the genome DNA extracting method according to claim 1 based on multigelation mode, which is characterized in that step
(6) in, the cold ethyl alcohol of the mixed solution 2 times of volumes of addition obtained to step (5) and the 3mol/L sodium acetate of 0.1 times of volume are water-soluble
Liquid is centrifuged, precipitating.
10. the genome DNA extracting method according to claim 1 based on multigelation mode, which is characterized in that step
(7) drying at room temperature after 70% ethanol washing is added in, in the precipitating that obtains to step (6), adds ddH2O or TE buffer solution
DNA。
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| CN110511977B (en) * | 2019-09-05 | 2023-07-21 | 武汉华大基因技术服务有限公司 | Genomic DNA extraction method, sequencing method and kit |
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