CN107418952A - A kind of extracting method of edaphon macro genome DNA and corresponding kit - Google Patents
A kind of extracting method of edaphon macro genome DNA and corresponding kit Download PDFInfo
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Abstract
The invention discloses a kind of extracting method of edaphon macro genome DNA and corresponding kit.The extracting method of edaphon macro genome DNA of the present invention is to carry out pre-treatment to pedotheque to be measured, effectively avoid the influence to lysozyme such as heavy metal, high salinity and low pH, again by adding lysozyme and combining CTAB and SDS collaborations cell lysis twice, further freeze thawing treatment crushes more microorganism walls, DNA molecular therein dissolution to greatest extent, it ensure that DNA abundance, fragment integrity and purity.Extracting method of the present invention is simple to operate, practical, and the DNA for extracting to obtain may be directly applied to solve the 26S Proteasome Structure and Function of analyzing soil microbial community.The extracting method of the present invention edaphon macro genome DNA extremely low especially suitable for extraction extreme environment, biomass, and corresponding DNA extraction kit can be made into according to extracting method of the present invention, have a good application prospect.
Description
Technical field
It is grand more specifically to a kind of edaphon the invention belongs to biological genome DNA extractive technique field
The extracting method of genomic DNA and corresponding kit.
Background technology
Extreme environment refers to thermal extremes, low temperature, high pressure, hyperoxia, high salt, height radiation, arid, Extreme acid or alkali
Property, the environment of the feature such as high content of beary metal.Typical extreme environment includes volcano, desert, hot spring, salt lake, submarine hydrothermal solution mouth,
Polar region etc..Situation of Microorganism Under Extremity Environment type is various, and distribution is very extensive on earth, and studying its vital movement has important reason
By research and actual application value.The method of microorganisms mainly has two kinds at present, i.e., traditional laboratory pure culture method and
Exempt from cultivation.Environment is survived a large amount of microorganisms, but educable microorganism only accounts for 1% or so, and most microorganism by
In various reasons, acquisition can not be cultivated.
Grand genome method avoids the problem of microorganism is separately cultured, increased by extracting total genome in sample
The chance of new species is found, can comprehensively and objectively reflect the diversity of microorganism in sample.With high throughput sequencing technologies
Fast development and sequencing cost significantly reduce, using metagenomics study extreme microorganism structure of community turn into it is micro-
One important content of biological and ecological research.The research object of metagenomics is whole microorganism total DNAs in specific environment, because
This DNA extractions are the successful committed steps of grand genome research, and high quality DNA acquisition is the basis of high-flux sequence.
DNA for the grand genome research of high-flux sequence needs to meet two conditions, should obtain as far as possible in sample
The gene of all microorganisms, ensure the integrity degree and DNA purity of fragment again.It is presently used for high-flux sequence or structure base
Because the genomic DNA in group library is typically using SDS methods (neopelex), CTAB methods (cetyl trimethyl bromination
Ammonium) or extract the methods of absorption centrifugal column based on silica matrix.In the market also has many commercial genome extracts kits all
It can efficiently extract bacterial genomes DNA, but these kits are both for educable bacterium, and it is micro- for exempting to cultivate
Biologicak efficiency is then very low.Because content of microorganisms is few under extreme environmental conditions, and particularly some contain a large amount of salt from
The serious puzzlement DNA such as son, extreme acid alkalescence condition extraction.Extracted using conventional DNA extraction method or general reagent box
Macro genome DNA efficiency is low, and abundance is not good enough, can not obtain purity height, the macro genome DNA of Functionality, quality and appealing design.
The content of the invention
It is an object of the invention to：Existing extraction when overcoming existing conventional extreme environment soil microbial DNA extraction
Efficiency is low, can not meet the problems such as sequencing requires, there is provided a kind of extraction of quick, easy edaphon macro genome DNA
Method, the DNA that this method is extracted to obtain have the advantages that high abundance, high concentration, high-purity, impurity are few, are easy to subsequent high pass amount
Analysis.
In order to realize foregoing invention purpose, the present invention provides a kind of extracting method of edaphon macro genome DNA, its
Comprise the following steps：
(1) it is 2 by volume ratio:3~3:2 pedotheque to be measured mixes with buffer A, 50~55 DEG C be incubated 15~
After supernatant is removed in 20min, centrifugation, add buffer A and 37 DEG C of lysozyme incubation 30min~1h, then through 20mg/ml Proteinase Ks with
37 DEG C of 20~30min of processing of CTAB, then add the SDS that concentration is 20%, 65~68 DEG C of 1~2h of cell lysis, and centrifugation obtains
DNA supernatant and precipitation must be contained；
(2) SDS mixing, 65~68 DEG C of water-baths of buffer B and concentration for 20% are added in being precipitated obtained by step (1)
15~20min then -80 DEG C freezing 10~15min, multigelation three times after, centrifuging and taking supernatant, with step (1) obtained by supernatant
Liquid mixes；The volume ratio of precipitation obtained by step (1) and buffer B is 1:3~3:2；The SDS and buffer B that concentration is 20%
Volume ratio is 1:1~9；
(3) isometric phenol is added:Chloroform:Isoamyl alcohol extraction albumen, then carry out 1~2h of precipitation with isopropanol；Will be mixed
Close liquid to be added in centrifugal purification post, liquid, the as grand base of edaphon in centrifuge tube are collected after centrifuge washing TE elutions
Because of a group DNA solution；
Wherein, the pH of the buffer A is 8.0, including 0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M
NaH2PO4With 0.1M Na2HPO4；
The pH of the buffer B is 8.0, including 0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M
NaH2PO4、0.1M Na2HPO4And 1wt%CTAB.
As a kind of optimal technical scheme of the extracting method of edaphon macro genome DNA of the present invention, above-mentioned soil
The extracting method of microorganism macro genome DNA, it comprises the following steps：
(1) it is 3 by volume ratio:2 pedotheque to be measured mixes with buffer A, supernatant is removed in 55 DEG C of incubation 20min, centrifugations
Afterwards, 37 DEG C of incubation 30min~1h of buffer A and lysozyme are added, then through the 37 DEG C of processing of 20mg/ml Proteinase Ks and CTAB
20min, then adds SDS, 65 DEG C of 1~2h of cell lysis that concentration is 20%, and centrifugation obtains supernatant and precipitation containing DNA；
(2) buffer B is added in precipitating obtained by the step (1) and SDS mixing that concentration is 20%, 65 DEG C of water-bath 15min
Then -80 DEG C freezing 15min, multigelation three times after, centrifuging and taking supernatant, with step (1) obtained by supernatant mix；Step
(1) volume ratio of gained precipitation and buffer B is 1:3, the SDS and the volume ratio of buffer B that concentration is 20% are 1:5；
(3) isometric phenol is added:Chloroform:Isoamyl alcohol extraction albumen, then isopropanol is subjected to precipitation 1h；By mixed liquor
It is added in centrifugal purification post, liquid, the as grand genome of edaphon in centrifuge tube is collected after centrifuge washing TE elutions
DNA solution；
As a kind of optimal technical scheme of the extracting method of edaphon macro genome DNA of the present invention, step (1)
In, described addition 37 DEG C of incubation times of buffer A and lysozyme are 1h.
As a kind of optimal technical scheme of the extracting method of edaphon macro genome DNA of the present invention, step (1)
In, the time of the cell lysis is 2h.
As a kind of optimal technical scheme of the extracting method of edaphon macro genome DNA of the present invention, step (1)
In, in described 37 DEG C of processing 20min through 20mg/ml Proteinase Ks and CTAB, every 5~10min is reverse to be mixed once.
As a kind of optimal technical scheme of the extracting method of edaphon macro genome DNA of the present invention, step (3)
In, the number of the TE elutions is multiple.
As a kind of more preferably technical scheme of the extracting method of edaphon macro genome DNA of the present invention, step (3)
In, the number of the TE elutions is 3 times.
The extracting method of edaphon macro genome DNA of the present invention is applicable not only to normal soil sample, also especially suitable
Pedotheque for the extremely low biomass of extreme environment is tested.
In order to realize foregoing invention purpose, present invention also offers a kind of extraction of edaphon macro genome DNA examination
Agent box, it includes buffer A, buffer B, the lysozyme soln that pH 8.0, concentration are 50mg/ml, Proteinase K, 10wt%
CTAB, 20wt% SDS, 5M NaCl, 2mM EDTA, 1.2 volume % Tritonx-100 isopropanols, TE buffer solutions and 70
Volume % ethanol solution；
Wherein, the pH of the buffer A is 8.0, including 0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M
NaH2PO4With 0.1M Na2HPO4；
The pH of the buffer B is 8.0, including 0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M
NaH2PO4、0.1M Na2HPO4And 1wt%CTAB.
The extracts kit of the above-mentioned edaphon macro genome DNA of the present invention can be not only used for extracting in normal soil sample
Microorganism macro genome DNA, be also particularly suitable extraction the extremely low biomass of extreme environment pedotheque in microorganism it is grand
Genomic DNA.
Compared with prior art, the present invention has the advantages that：
(1) there is the defects of impurity is more, easily causes genome loses in manual extraction method, and extracting method of the present invention is by hand
Extraction (CTAB+SDS cracking) extracts the mode that is combined with kit, and the adsorption column in kit can be with efficient absorption DNA, more
The deficiency that genome caused by having mended manual extraction method loses, both can disposably obtain larger amount of DNA extracts, again can be effective
Improve genome yield；
(2) extracting method of the present invention first passes through the pre-treatment step centrifuged after specific buffer solution is incubated, can suppress DNA's
Degraded, chelates excessive metal ion, because the presence of metal ion can influence the activity of lysozyme, removes metal ion
The effect of lysozyme can be played to greatest extent, improve the recovery rate of genome；
(3) extracting method of the present invention is by increasing sample size from soil (extreme environment soil) by lysozyme broken wall,
Reach maximum through Proteinase K and the mode of CTAB+SDS collaborations cracking and thawing to obtain the crude extract containing more DNA again
Limit obtains the purpose of genome；
(4) in extracting method of the present invention, using multiple elution, maximum possible is obtained compared with high DNA yield, and can be according to elution
The quantity regulation DNA concentration of liquid, detection indicate that (Fig. 1, table 1), the sand macro genome DNA matter that extracting method of the present invention obtains
Amount is higher, and purity is good.
(4) extracting method of the present invention ensure that DNA abundance, fragment integrity and purity, have simple to operate, practicality
The characteristics of strong, the DNA for extracting to obtain may be directly applied to solve the 26S Proteasome Structure and Function of analyzing soil microbial community.
Brief description of the drawings
With reference to the accompanying drawings and detailed description, the present invention and beneficial effect are described in detail.
Fig. 1 is the extraction grand genome of sand of the embodiment of the present invention and other SDS added column method and CTAB/SDS to centrifuge
Method collection method extracts the comparison diagram of DNA electrophoresis.
Embodiment
In order that goal of the invention, technical scheme and the advantageous effects of the present invention become apparent from, with reference to embodiments,
The present invention will be described in further detail.It should be appreciated that the embodiment described in this specification is just for the sake of explanation
The present invention, being not intended to limit the present invention, the formula of embodiment, ratio etc. can suit measures to local conditions to make a choice and have no reality to result
Matter influences.
Embodiment 1
1. reagent configures
(1) buffer A：0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M NaH2PO4And 0.1M
Na2HPO4, pH is tuned into 8.0, and deionized water constant volume is into 1L, autoclave sterilization.
(2) 50mg/ml lysozymes：20mM Tris-HCl, 2mM EDTA, volume fraction are 1.2% Triton x-
100。
(3) 10wt%CTAB:1gCTAB powder is weighed, 100ml (needing 65 DEG C of heating water baths to dissolve) is settled to sterilized water.
(4) buffer B：0.5g CTAB are weighed, 50ml (needing 65 DEG C of heating water baths to dissolve) is settled to buffer A.
(5) 20%SDS：10g SDS are weighed, being settled to 50ml with sterilized water (needs 68 DEG C of heating water baths to dissolve, pH is tuned into
7.2)。
(6) phenol：Chloroform：Isoamyl alcohol (25：24：1).
(7) chloroform：Isoamyl alcohol (24：1).
(8) isopropanol (4 DEG C) precooling, 70% ethanol.
(9)RNase-free water。
(10)OMEGA Bacterial DNA Kit。
(11) Proteinase K 20mg/ml.
2. sample pre-treatments
(1) 15g sands are taken in 50ml centrifuge tubes, add 30ml buffer As, the mixing 10min that is vortexed (is mixed into homogenate shape
There is no any particle or lump adherent), 55 DEG C of water-bath 20min；
(2) 10000xg, 10min, supernatant discarding are centrifuged (precipitation can put -80 DEG C if do not continued with)
3.DNA is extracted
(1) precipitation after handling adds 12ml buffer As, adds 500 μ l lysozymes, 37 DEG C of water-bath 30min~1h
(period gently overturns every 5~10min and mixed).
(2) add 100 μ L Proteinase Ks (20mg/mL) and 1.5ml 10%CTAB gently to mix, gently mix, 37 DEG C of incubations
20min (every 5~10min is reverse to be mixed).
(3) 1.5ml 20%SDS are added, (every 5~10min turns upside down mixings, transfer supernatant by 65 DEG C of 1~2h of water-bath
Into new centrifuge tube (50ml, sterilizing, be careful not to encounter white precipitate).
(4) soil precipitation adds 4.5ml buffer Bs and 0.5ml 20%SDS, is vortexed and mixes 20s, 65 DEG C of water-baths
15min, water-bath terminate rear multigelation (- 80 DEG C, 15min, 65 DEG C, 5min) room temperature 10000g centrifugations 10min three times.Take
Clearly, and above supernatant mixes twice, not encounter white precipitate.
(5) isometric phenol is added：Chloroform：Isoamyl alcohol (25：24：1), turn upside down and mix 3min.Claim together with pipe sleeve
Trim again, 10000g centrifugations 10min.
(6) supernatant adds isometric chloroform：Isoamyl alcohol (24：1), turn upside down mixing.Weighed trim together with pipe sleeve,
10000g centrifuges 10min.
(7) supernatant (50ml, sterilizing) into new centrifuge tube is shifted, adds the isopropanol precipitating DNA (4 of 0.6 times of volume
DEG C precooling), gently overturn and mix 1min, room temperature places 1~2h.
4. kits
(1) 700ml crosses OMEGA Bacterial DNA Kit pillars every time, centrifuges 30s, excessively complete to whole.
(2) post will be collected to be transferred in the centrifuge tube that Kit is furnished with, toward adding 500 μ lHB Buffer in pillar, normal temperature from
Scheming centrifugation 1min (10000g).
(3) filtrate is removed, 700 μ l DNA Wash Buffer, normal temperature centrifuge 1min is added toward pillar is interior
(10000g)。
(4) previous step is repeated once.
(5) filtrate is removed, normal temperature centrifuge sky gets rid of 2min (13000g).
(6) by posts transfer into sterile centrifugation tube (1.5ml), it is placed in super-clean bench and air-dries (5min).
(7) to adding RNase-free water dissolving DNAs (the 50 μ l every time, after elution that preheat in right amount on pillar inner membrance
Lower concentration is first surveyed, continues to add 50 μ l if higher, if low, takes filtered solution after a post) stand 3~5min, normal temperature centrifugation
Machine centrifugation 2min (13000g) at least crosses pillar three times.
Embodiment 2 utilizes the SDS methods extraction grand genome of sand
(1) 15g sands are taken in 50ml centrifuge tubes, add 30ml buffer As, is vortexed and mixes, 55 DEG C of water-bath 20min；
(2) 9000g, 10min, supernatant discarding are centrifuged；
(3) 10ml buffer As are added into centrifuge tube, use freeze-thaw method smudge cells：- 80 DEG C of 15min -65 DEG C water-baths
5min (is repeated 3 times, mixed during 65 DEG C of warm bath without reverse).
(4) room temperature cooling (~10min), into centrifuge tube adding 1ml lysozymes (10mg/ml), (final concentration is about
0.5mg/ml), 37 DEG C of water-bath 1h (period gently overturns every 10min and mixed).
(5) (final concentration is about 100 μ for addition 0.5ml 10%SDS (final concentration is about 0.5%SDS) and 20 μ l Proteinase Ks
G/ml), 58 DEG C of water-bath 1h (mixing of being turned upside down per 10min, last 4 DEG C of centrifuges of 20min precoolings).
(6) (4000~6000) xg centrifuges 7~10min.
(7) supernatant (50ml, sterilizing, be careful not to encounter white precipitate) into new centrifuge tube is shifted.
(8) remaining soil precipitation adds 4.5ml buffer Bs and 0.5ml 20%SDS, is vortexed and mixes 20s, 65 DEG C of water
Bathe 15min, room temperature 10000g centrifugations 10min.Take supernatant, and an above supernatant mixing.
(9) isometric phenol is added in supernatant：Chloroform：Isoamyl alcohol (25：24：1), turn upside down mixing.10000xg from
Heart 15min.
(10) supernatant (50ml, sterilizing) into new centrifuge tube is shifted, adds isometric chloroform：Isoamyl alcohol (24：
1), turn upside down mixing.10000xg centrifuges 15min.
(11) supernatant (50ml, sterilizing) into new centrifuge tube is shifted, adds (4 DEG C of isometric isopropanol precipitating DNA
Precooling), mixing gently, room temperature places 1h.
(12) 600ml crosses post with the pillar in OMEGA Bacterial DNA Kit every time, centrifuges 1min.
(13) post will be collected to be transferred in the centrifuge tube that Kit is furnished with, toward adding 500 μ lHB Buffer in pillar, normal temperature from
Scheming centrifugation 1min (10000g).
(14) filtrate is removed, 700 μ l DNA Wash Buffer, normal temperature centrifuge 1min is added toward pillar is interior
(10000g)。
(15) previous step is repeated once.
(16) filtrate is removed, normal temperature centrifuge sky gets rid of 2min (13000g).
(17) by posts transfer into sterile centrifugation tube (1.5ml), it is placed in super-clean bench and air-dries (5min).
(18) to adding RNase-free water (50-100 μ l) dissolving DNA for preheating in right amount, normal temperature on pillar inner membrance
Centrifuge 2min (13000g)
Embodiment 3 extracts the grand genome of sand using the method that is collected by centrifugation
(1) 15g sand soil samples are claimed, to the grinding alms bowl of Liquid nitrogen precooler.
(2) liquid nitrogen grinding is added, continues to add liquid nitrogen grinding before sample dissolving, repeats plus grinds three times three times
(3) all samples are gone to 50-ml Falcon pipes plus 16.5ml DNA extracts.Mix and (survey the pH of lower supernatant such as
Fruit pH is not 8, is transferred to 8.
(4) plus 40 μ l Proteinase Ks (20mg/ml), gentle overturn mix, 37 DEG C, 30min.
(5) 65 DEG C ,+1.83ml 20%SDS, mix, incubate 1~2h.Mixed once per 20min.
(6) 7000rpm, 15min, supernatant is collected to new Fecon tube, rear 4 DEG C of storage.
(7) add 6ml extracts, add 0.67ml 20%SDS, mix, 65 DEG C, 15min (the remaining precipitation samples of extracting again
Product).
(8) 7000rpm 15min, the supernatant obtained twice is mixed.
(9)+1x volume (~22ml) isoamyl alcohol：Chloroform (1:24=20ml isoamyl alcohol+480ml
Chloroform, d=1.48g/cm3), shake 5min and mix extracting (~24).
(10) 7000rpm 15min, collect supernatant and managed to new Falcon.
(11) isoamyl alcohol：Chloroform extracts once again, collects supernatant to round bottom centrifuge tube, record lower volume (~19 ml).
(12) accurately add 0.6 volume isopropanol (2-propanol, isopropanol, FW 60.10), turn upside down mixed
It is even, stand 1h.
(13)10500rpm 45min.Careful transfer supernatant is managed to new Falcon, stays (1-1.5ml) supernatant.
(14) residual supernatant (1-1.5ml, containing DNA) is collected to a 1.5ml centrifuge tube.
(15) 13000rpm 5-8min enrichment DNAs (wash the big centrifuge tube region of DNA domain part (0.5ml) of round bottom to close with cleaning
And arrive 1.5ml vial).
(16) plus 70% cold ethanol of+1ml washings DNA is precipitated, and 13000rpm, 5min, carefully removes supernatant.
(17) in the drying precipitated 30min of ventilating kitchen.
(18) 20 μ l H are added2O dissolving DNAs, tube wall is rapped, dissolve and somewhat centrifuge, 4 DEG C of overnight dissolving DNAs.
As a result
Fig. 1 is referred to, Fig. 1 is the sand DNA electrophoretograms that distinct methods obtain, wherein, M is DNA Marker (15000),
1 is the DNA that embodiment 3 is collected, and 2 be the DNA that embodiment 2 is collected, and 3 be the DNA that embodiment the inventive method is collected.
The sand DNA concentration contrast that distinct methods obtain refers to table 1.
The sand DNA concentration contrast that the distinct methods of table 1 obtain
| Method | CTAB+SDS cracks+cross post | SDS cracks+cross post | CTAB+SDS is cracked+is collected by centrifugation |
| Concentration ng/ μ l | 117.5 | 67.4 | 90.4 |
| 260/280 | 1.86 | 1.75 | 1.65 |
| 260/230 | 1.73 | 1.04 | 1.20 |
Such as Fig. 1, DNA Normal Agarose Gel images are extracted from 3 kinds of methods, with existing other method phase
Than macro genome DNA (at Fig. 1 marks 3) band that the inventive method obtains is more bright, clearly, completely, and without degraded, says
The DNA better quality that bright the inventive method obtains, concentration are higher.Although the method for embodiment 2 obtain DNA also have higher brightness but
It is to have degraded, hangover is serious.Embodiment 3 without degraded but band brightness it is weaker, DNA content is few.Moreover, from Nano Drop
Spectrophotometer spectrophotometers testing result (table 1) sees that the DNA concentration that this method obtains is significantly higher than its other party
Method, and 260/280 and 260/230 ratio is also higher than other method, illustrates that this method obtains DNA protein, carbohydrate, salt etc.
Impurity content is few, therefore purity is more preferable；To sum up, the macro genome DNA that the inventive method obtains have higher concentration and purity with
And more preferable integrality, the requirement of grand gene order-checking can be better met.
Above-listed detailed description is illustrating for one of present invention possible embodiments, and the embodiment simultaneously is not used to limit
The scope of the claims of the present invention, all equivalence enforcements or change without departing from carried out by the present invention, it is intended to be limited solely by the scope of the claims of this case
In.
Claims (10)
1. a kind of extracting method of edaphon macro genome DNA, it is characterised in that comprise the following steps：
(1) it is 2 by volume ratio:3~3:2 pedotheque to be measured mixes with buffer A, 50~55 DEG C be incubated 15~20min, from
After the heart removes supernatant, 37 DEG C of incubation 30min~1h of buffer A and lysozyme are added, then through 37 DEG C of 20mg/ml Proteinase Ks and CTAB
20~30min is handled, then adds SDS, 65~68 DEG C of 1~2h of cell lysis that concentration is 20%, centrifugation is obtained containing the upper of DNA
Clear liquid and precipitation；
(2) buffer B is added in precipitating obtained by the step (1) and SDS mixing that concentration is 20%, 65~68 DEG C of water-baths 15~
20min then -80 DEG C freezing 10~15min, multigelation three times after, centrifuging and taking supernatant, with step (1) obtained by supernatant mix
Close；The volume ratio of precipitation obtained by step (1) and buffer B is 1:3~3:2；Concentration is 20% SDS and the volume of buffer B
Than for 1:1~9；
(3) isometric phenol is added:Chloroform:Isoamyl alcohol extraction albumen, then carry out 1~2h of precipitation with isopropanol；By mixed liquor
It is added in centrifugal purification post, liquid, the as grand genome of edaphon in centrifuge tube is collected after centrifuge washing TE elutions
DNA solution；
Wherein, the pH of the buffer A is 8.0, including 0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M
NaH2PO4With 0.1M Na2HPO4；
The pH of the buffer B is 8.0, including 0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M NaH2PO4、
0.1M Na2HPO4And 1wt%CTAB.
2. the extracting method of edaphon macro genome DNA according to claim 1, it is characterised in that including as follows
Step：
(1) it is 3 by volume ratio:After 2 pedotheque to be measured mixes with buffer A, supernatant is removed in 55 DEG C of incubation 20min, centrifugations, add
Enter 37 DEG C of incubation 30min~1h of buffer A and lysozyme, then 20min are handled through 37 DEG C of 20mg/ml Proteinase Ks and CTAB, so
SDS, the 65 DEG C of 1~2h of cell lysis that concentration is 20% are added afterwards, and centrifugation obtains supernatant and precipitation containing DNA；
(2) buffer B is added in precipitating obtained by the step (1) and SDS mixing that concentration is 20%, 65 DEG C of water-bath 15min then-
80 DEG C freezing 15min, multigelation three times after, centrifuging and taking supernatant, with step (1) obtained by supernatant mix；Obtained by step (1)
The volume ratio of precipitation and buffer B is 1:3, the SDS and the volume ratio of buffer B that concentration is 20% are 1:5；
(3) isometric phenol is added:Chloroform:Isoamyl alcohol extraction albumen, then isopropanol is subjected to precipitation 1h；Mixed liquor is added
Into centrifugal purification post, liquid in centrifuge tube is collected after centrifuge washing TE elutions, as edaphon macro genome DNA is molten
Liquid.
3. the extracting method of edaphon macro genome DNA according to claim 2, it is characterised in that in step (1),
Described addition 37 DEG C of incubation times of buffer A and lysozyme are 1h.
4. the extracting method of edaphon macro genome DNA according to claim 2, it is characterised in that in step (1),
The time of the cell lysis is 2h.
5. the extracting method of edaphon macro genome DNA according to claim 2, it is characterised in that in step (1),
In described 37 DEG C of processing 20min through 20mg/ml Proteinase Ks and CTAB, every 5~10min is reverse to be mixed once.
6. the extracting method of edaphon macro genome DNA according to claim 2, it is characterised in that in step (3),
The number of the TE elutions is multiple.
7. the extracting method of edaphon macro genome DNA according to claim 6, it is characterised in that in step (3),
The number of the TE elutions is 3 times.
8. according to the extracting method of edaphon macro genome DNA described in any one claim in claim 1~7,
Characterized in that, the soil is the soil of the extremely low biomass of extreme environment.
9. a kind of extracts kit of edaphon macro genome DNA, it is characterised in that including buffer A, buffer B, pH
The lysozyme soln for being 50mg/ml for 8.0, concentration, Proteinase K, 10wt%CTAB, 20wt% SDS, 5M NaCl, 2mM
EDTA, 1.2 volume % Triton x-100 isopropanols, TE buffer solutions and 70 volume % ethanol solution；
Wherein, the pH of the buffer A is 8.0, including 0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M
NaH2PO4With 0.1M Na2HPO4；
The pH of the buffer B is 8.0, including 0.1M EDTA, 0.1M Tris-HCl, 1.5M NaCl, 0.1M NaH2PO4、
0.1M Na2HPO4And 1wt%CTAB.
10. the extracts kit of edaphon macro genome DNA according to claim 8, it is characterised in that the soil
Earth is the soil of the extremely low biomass of extreme environment.
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