CN106399297A - Method for economically and rapidly extracting microbial genome DNA in fermented grains - Google Patents
Method for economically and rapidly extracting microbial genome DNA in fermented grains Download PDFInfo
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Abstract
The invention relates to the technical field of brewing of Baijiu, and in particular to extraction of microbial genome DNA in fermented grains. The method comprises the following steps: (1) wall breaking of microbial cells in the fermented grains: weighing a fermented grain sample and quartz sand, adding lysate, and conducting shaking centrifugation so as to obtain a coarse nucleic acid extracting solution; and (2) nucleic acid purification: adding a mixture of phenol: chloroform: isoamylol (P.C.I), conducting centrifugation to remove protein, adding a silica gel membrane binding solution, conducting filtering by virtue of a nucleic acid purification column, eluting the nucleic acid purification column by virtue of 70% ethanol, adding de-ionized water or a TE solution and preserving the DNA. The method is simple and convenient to operate, low in cost consumption, low in labor intensity and time-saving, and the extracted microbial genome in the fermented grains is high in quality and is applicable to such molecular biology analysis as next generation sequencing, fluorescent quantitative PCR and the like.
Description
Technical field
The invention belongs to brewing technical field is and in particular in one of liquor-making enterprises economic rapid extraction fermented grain
The method of microbe genome DNA.
Background technology
In fermented grain, microbe genome DNA extracts is microorganism composition in researching white spirit brewing process, microbial function, micro-
Biological metabolism and the necessary condition of microorganism and environmental concerns.Therefore, the extraction rate of fermented grain genomic DNA, extraction quality exist
Play an important role in liquor production and research.
At present, method microbe genome DNA in fermented grain extracted can be divided into portable and RNA isolation kit.Lifting manipulation adopts
Liquid nitrogen grinding broken wall or enzymolysis broken wall, phenol：Chloroform：Isoamyl alcohol (P.C.I) purifies, isopropanol or ethanol precipitation DNA, water-soluble
Solution DNA.Although the method low cost, the DNA take longer, high labor intensive, extracting is of low quality.RNA isolation kit is using joining
The reagent put, only need to be by flow operations.Although the method is simple to operate, because in fermented grain, micro organism quantity is relatively
Few, 50mL specification kit absorption column bleed is larger, and the DAN concentration extracting is very low, 2mL specification kit take longer,
High labor intensive；And equally exist the low problem of concentration, need to merge the DNA of the sample of multiple repetitions, lead to cost intensive,
Be not suitable for the conventional detection of enterprise.
Therefore, the method that this area needs genomic DNA in a kind of economic rapid extraction fermented grain.
Content of the invention
In order to solve the problems, such as fermented grain extracting genome DNA in prior art, in liquor production research and development, for warp
Microbe genome DNA in Ji rapid extraction fermented grain, the invention provides a kind of economic fast and high quality extracts micro- life in fermented grain
The method of thing genomic DNA.The method combines portable and kit method, existing method is improved and optimizated, considers extraction
Cost, ease-to-operate, labour and extraction quality etc..By comparing checking, develop micro- in a kind of economic rapid extraction fermented grain
The method of biological genome DNA.The method is easy and simple to handle, Expenses Cost is low, labour intensity is little, save the time, extract fermented grain
Middle microbial genome quality is high.
Realize especially by technical scheme below：
In a kind of economic rapid extraction fermented grain, the method for microbe genome DNA, comprises the following steps：
1) microbial cell broken wall in fermented grain：Grinding agent, fermented grain sample and lysate three are mixed, shakes at a high speed, so
After be centrifuged；
Described grinding agent is bead, quartz sand, aluminum oxide；Preferably, it is quartz sand；It is highly preferred that being 12-20 mesh
Quartz sand；It is highly preferred that the quartz sand for 16 mesh.
2) nucleic acid purification.
In the preferred embodiment of the invention, selected grinding agent quartz sand.Quartz sand flintiness is high, have corner angle, glass
Glass bead surface smooths, and in same time, quartz sand crushing effect is better than bead；Secondly, fermented grain sample DNA grinds needed for extracting
Grinding agent is more, and quartz sand cost is less than bead.The species of grinding agent and particle diameter have multiple, in multiple particle diameters disclosed by the invention
Bead and quartzite sand grind agent in, the quartz sand taking 12-20 mesh, as grinding agent, can extract the gene of high concentration
Group DNA.
Wherein, step 1) in,
The ratio of the three of grinding agent, fermented grain sample and lysate is 15～22g:6～9g:16～24mL；Preferably, it is
18.5g:7.5g:20mL；
Cracking formula of liquid is as follows：1.5-2.5%CTAB (w/v), 90-112mM Tris-HCl (pH 8.0), 18-24mM
EDTA (pH8.0), 1.2-1.6M NaCl；Preferably 2%CTAB (w/v), 100mM Tris-HCl (pH 8.0), 20mM EDTA
(pH8.0), 1.4M NaCl；
Centrifuge RPMs are 10000rpm, and centrifugation time is 10min.
Step 2) in, the purifying of nucleic acid specifically comprises the steps of：
A) removal of impurities：Take step 1) in centrifugation after supernatant add P.C.I, mix, centrifugation；
Described P.C.I is 24-26 volume of phenol：23-25 volume of chloroform：0.5-1.5 volume isoamyl alcohol；Preferably 25 volumes
Phenol：24 volume of chloroform：1 volume isoamyl alcohol；
P.C.I is 1 with the volume ratio of supernatant:0.8～1.2；
Centrifuge RPMs are 10000rpm, and centrifugation time is 20min.
B) adsorb：Take step be a) in supernatant be combined liquid with pellosil and mix, then cross nucleic acid purification post, centrifugation abandons
Filtrate；Described pellosil combines the mixed liquor that liquid is that pH is the guanidine hydrochloride of 4-6.5 and potassium acetate.
In the preferred embodiment of the invention, guanidine hydrochloride and potassium acetate that described pellosil is 5 with reference to liquid for pH mixed
Close liquid.
In the preferred embodiment of the invention, the nucleic acid purification post of selection is the nucleic acid purification post of 15mL；It is highly preferred that institute
The nucleic acid purification post stated contains 8 layers or more of pellosil.When the number of plies of pellosil reaches some, DNA can stablize
Be attached on pellosil, wash later and difficult for drop-off in the step of post get off.Described nucleic acid purification post can select commercially available
Nucleic acid purification post.
The volume ratio that supernatant is combined liquid with pellosil is 1:1.5～2.5；
Pellosil with reference to liquid is：3-4M guanidine hydrochloride：0.5M potassium acetate；Preferably 4M guanidine hydrochloride：0.5M potassium acetate.
C) wash post：2.5-3.5mL 70% ethanol is added to cross nucleic acid purification post, then high speed centrifugation dries nucleic acid purification post,
It is dried under natural conditions；
The number of times washing post is twice；
The time spontaneously drying is 30～60min；Preferably, it is 40min.
D) elute：Eluent dissolving DNA, the lower DNA solution of centrifugation wash-out are added on the pellosil of nucleic acid extraction column.
Described eluent is selected from sterilized water or TE solution；
The volume of eluent is 50～300 μ L；
Centrifuge RPMs are 10000rpm, and centrifugation time is 2min.
In a kind of economic rapid extraction fermented grain, the method for microbe genome DNA is it is characterised in that more specifically step is such as
Under：
After the quartz sand sterilizing, drying of 12～20 mesh, weigh 15～22g and load in 50mL sterile centrifugation tube；
Weigh 6～9g fermented grain sample in centrifuge tube, add 16～24mL lysate, homogeneous instrument shakes 1min, 10000rpm
Centrifugation 10min；
Transfer supernatant, to 50mL sterile centrifugation tube, plus 0.8～P of 1.2mL:C:I, shakes up, and 11000rpm is centrifuged
20min；Described P.C.I is 25 volume of phenol：24 volume of chloroform：1 volume isoamyl alcohol.
Transfer supernatant, to 50mL sterile centrifugation tube, plus 1.5～pellosil of 2.5mL combines liquid, shakes up；Use 15mL nucleic acid
Purification column filters, and abandons filtrate；Pellosil with reference to liquid for pH be 5 guanidine hydrochloride and potassium acetate mixed liquor.
Jia 2.5 in adsorption column～3.5mL 70% ethanol, filter, abandon filtrate, be repeated once.
10000rpm blank pipe is centrifuged 2min, abandons filtrate；
1h is dried under purification column natural conditions, adds 50～300 μ L aseptic deionized waters or TE solution dissolving DNA, filter,
Filtrate is loaded in 1.5mL sterile centrifugation tube, and -20 DEG C save backup.
Described cracking formula of liquid is as follows：2%CTAB (w/v), 100mM Tris-HCl (pH 8.0), 20mM EDTA
(PH8.0), 1.4M NaCl.
It is as follows that described pellosil combines formula of liquid：4M guanidine hydrochloride：0.5M potassium acetate.
Described step 15mL nucleic acid purification post contains 8 layers of pellosil.
Inventor finds, the selection of specific grinding agent can affect to purify Column methods application to a great extent
The extraction effect extracting in fermented grain microbial DNA.In multiple amount of abrasive, the quartz sand of certain mesh number presents prominent fitting
Answer sexual clorminance.
Compared with prior art, what the present invention obtained has the beneficial effect that：
1. quick：Liquid nitrogen grinding broken wall needs 30min, and expends muscle power；Enzymolysis broken wall at least needs 2h；This method only needs height
Speed concussion 1min, you can make clasmatosis, discharge nucleic acid.The time that whole DNA extracts is 2～2.5h, and liquid nitrogen grinding need 4～
4.5h, enzymolysis broken wall needs 4.5～5h.
2. low cost：RNA isolation kit extracts a sample and needs 300 yuan about costs, and cost control can be existed by this method
Within 50 yuan, the method is easy and simple to handle, Expenses Cost is low, labour intensity is little.
3.DNA mass is high：Ethanol or isopropanol precipitating nuclei aoid methods are adopted, nucleic acid impurities are more in lifting manipulation, one
As need add purification step；This method adopts pellosil adsorption of DNA, need not purify, and extracts genomic DNA quality high.This method
The genomic DNA quality extracted is suitable with RNA isolation kit.
4. the present invention adopts the grinding agent as broken wall for the quartz sand of specific mesh number, in the DNA of fermented grain microorganism extracts
Get non-obvious effect.The broken wall that in prior art, fermented grain DNA is extracted is liquid nitrogen or enzymatic isolation method, and above two method is taken
When laborious；Or employ in DNA extraction kit bead as grinding agent, however, bead is as the grinding of the present invention
During agent, because surface smooths, in same time, shell-broken effect is poorer than quartz sand；Secondly, needed for this experiment, grinding agent is more, stone
The cost of sand is far below bead.
To sum up, the present invention adopts the method that lifting manipulation and kit combine, little in time-consuming and cost premise, permissible
Ensure that the quality of DNA extraction and concentration substantially achieve and adopts RNA isolation kit, be conducive on a large scale should in liquor-making enterprises
The method extracted with this fermented grain microbial DNA.
Brief description
The agarose gel electrophoresis that Fig. 1 extracts microbe genome DNA in fermented grain for the present invention becomes phasor.
Fig. 2 extracts the concentration mensuration of microbe genome DNA in fermented grain for the present invention.
The grinding agent shell-broken effects comparison agarose gel electrophoresis that Fig. 3 is different becomes phasor.
Fig. 4 is the concentration mensuration that comparative example 1 extracts microbe genome DNA in fermented grain.
Fig. 5 is the concentration mensuration that comparative example 2 extracts microbe genome DNA in fermented grain.
Specific embodiment
Further illustrate technical scheme below by way of specific embodiment, specific embodiment does not represent to this
The restriction of bright protection domain.Some nonessential modifications that other people are made according to theory of the present invention and adjustment still fall within this
Bright protection domain.
Operating procedure in the following example is taking in the operation of 50mL centrifuge tube as a example.As needed other scales to extract, in proportion
Zoom in or out reagent and sample size.
The configuration of two kinds of reagent
1. lysate
Lysate component (CTAB)
2%CTAB (w/v), 100mM Tris-HCl (pH 8.0), 20mM EDTA (pH8.0), 1.4M NaCl.
Cracking liquid and preparation method thereof
Configuration Tris-HCl, pH are adjusted to 8.0, sterilizing；Configuration EDTA, pH are adjusted to 8.0,
Sterilizing；After four kinds of component mixing, sterilizing.
2. pellosil combines liquid
(1) pellosil combines liquid component
4M guanidine hydrochloride, 0.5M potassium acetate (pH 5.0).
(2) pellosil combines liquid and preparation method thereof
After two kinds of component mixing, with concentrated hydrochloric acid, pH is adjusted to 5.0, sterilizing.
In embodiment 1 fermented grain, microbe genome DNA extracts
1. load after the quartz sand sterilizing, drying of 16 mesh, weighing 18.5g in 50mL sterile centrifugation tube.
2. weigh 7.5g fermented grain sample in centrifuge tube, add 20mLCTAB extract, homogeneous instrument shakes 1min, 10000rpm
Centrifugation 10min..
3. transfer supernatant, to 50mL sterile centrifugation tube, plus 0.8～1.2 times of volumes P:C:I (phenol：Chloroform：Isoamyl alcohol=
25:24:1), shake up, 11000rpm is centrifuged 20min.
4. transfer supernatant, to 50mL sterile centrifugation tube, plus 1.5～2.5 times of volume silica gel film combination liquid, is shaken up；Use 15mL
Adsorption column filters, and abandons filtrate, till having filtered.
5. add 3mL 70% ethanol in adsorption column, filter, abandon filtrate.
6. repeat step 5 is once.
7.10000rpm blank pipe is centrifuged 2min, abandons filtrate.
8. 1h is dried under adsorption column natural conditions, adds 300 μ L sterilized water dissolving DNAs, filter, filtrate is loaded on 1.5mL no
In bacterium centrifuge tube, -20 DEG C save backup.
In embodiment 2 fermented grain, microbe genome DNA extracts
1. load after the quartz sand sterilizing, drying of 12 mesh, weighing 15g in 50mL sterile centrifugation tube.
2. weigh 6g fermented grain sample in centrifuge tube, add 16mLCTAB extract, homogeneous instrument shakes 1min, 10000rpm from
Heart 10min..
3. shift supernatant to 50mL sterile centrifugation tube, plus equal-volume P:C:I (phenol：Chloroform：Isoamyl alcohol=25:24:
1), shake up, 11000rpm is centrifuged 20min.
4. shift supernatant to 50mL sterile centrifugation tube, plus 2 times of volume silica gel film combination liquid, shake up；Use 15mL adsorption column
Filter, abandon filtrate, till having filtered.
5. add 2.5mL 70% ethanol in adsorption column, filter, abandon filtrate.
6. repeat step 5 is once.
7.10000rpm blank pipe is centrifuged 2min, abandons filtrate.
8. 1h is dried under adsorption column natural conditions, adds 50 μ L sterilized water dissolving DNAs, filter, it is aseptic that filtrate is loaded on 1.5mL
In centrifuge tube, -20 DEG C save backup.
In embodiment 3 fermented grain, microbe genome DNA extracts
1. load after the quartz sand sterilizing, drying of 20 mesh, weighing 22g in 50mL sterile centrifugation tube.
2. weigh 9g fermented grain sample in centrifuge tube, add 24mLCTAB extract, homogeneous instrument shakes 1min, 10000rpm from
Heart 10min..
3. shift supernatant to 50mL sterile centrifugation tube, plus equal-volume P:C:I (phenol：Chloroform：Isoamyl alcohol=25:24:
1), shake up, 11000rpm is centrifuged 20min.
4. shift supernatant to 50mL sterile centrifugation tube, plus 2 times of volume silica gel film combination liquid, shake up；Use 15mL adsorption column
Filter, abandon filtrate, till having filtered.
5. add 3.5mL 70% ethanol in adsorption column, filter, abandon filtrate.
6. repeat step 5 is once.
7.10000rpm blank pipe is centrifuged 2min, abandons filtrate.
8. 40min is dried under adsorption column natural conditions, adds 300 μ L sterilized water dissolving DNAs, filter, filtrate is loaded on 1.5mL
In sterile centrifugation tube, -20 DEG C save backup.
Comparative example 1 lifting manipulation extracts the DNA in fermented grain sample
Extracting method bibliography (Li Delin, Ao Zonghua, Deng Bo, etc. fermented grain microorganism total DNA Study on Extraction Method
[J]. brewing science and technology, 2014,1:013.), specific as follows：Take 7.5g fermented grain in mortar, add liquid nitrogen be fully ground, proceed to from
Heart pipe adds 15mL CTAB extract (2%CTAB, 5moL/L NaCl, 1moL/L Tris-HCl (pH8), 0.5moL/L
EDTA) and 300 μ L mercaptoethanols, 65 DEG C of constant temperature blending instruments vibrate 30min, add 75 μ L protease k (20mg/mL), 55 DEG C of perseverances
Warm blending instrument vibration 30min room temperature is centrifuged 10min with 5000r/min, collects supernatant.Add equal-volume phenol chloroform isoamyl alcohol
(25: 24: 1) extract 1 time, 10min is centrifuged with 12000r/min, take supernatant to add equal-volume chloroform isoamyl alcohol (24: 1)
12000r/min is centrifuged 5min, takes supernatant, is repeated 2 times, and adds the isopropanol of 0.6 volume precooling to precipitate 1h at -20 DEG C
Afterwards 10min is centrifuged with 12000r/min, carefully outwells liquid.Precipitation is washed for several times with 70%voL ethanol, and extraction obtains DNA and blows
TE dissolving, -20 DEG C of preservations are added after dry.
Comparative example 2 RNA isolation kit extracts the DNA in fermented grain sample
Extracting method is joinedSoil DNA IsoLation kit kit specification.Concrete steps are such as
Under：
1st, past15mL PowerBead Solution is added in Bead Tube.This combination is named asBead Solution Tubes.
2nd, not more than 10g sample is added to arriveIn Bead Solution Tubes.Quick vortex oscillation
1min mixes.
3rd, detect C1 solution.If precipitation, 60 DEG C of water-baths are to CL.Plus 1.2mL C1 solution arrives
In Bead Solution Tubes, quick vortex 30s mixes.
4th,Bead Tubes is fixed on vortex instrument adapter, maximum (top) speed vortex continuous oscillation
10min.
5th, room temperature 2500g centrifugation 3min.
6th, transfer supernatant is to a clean Collection Tube.
7th, add 5mL C2 solution in supernatant, be vortexed and mix 5s.4 DEG C of incubation 10min.
8th, room temperature 2500g centrifugation 4min.
9th, avoid precipitating globule, transfer supernatant is in a new collecting pipe
10th, add 4mL solution C3 in supernatant, be vortexed and mix 5s.4 DEG C of incubation 10min.
11st, room temperature 2500g centrifugation 4min
12nd, avoid precipitating globule, transfer supernatant is in a new collecting pipe.
13rd, C4 solution is first shaken up using front.Add 30mL C4 solution in supernatant, be vortexed and mix 5s.
14th, the supernatant obtaining from 13 steps is filled it up with Spin Filter, room temperature 2500g is centrifuged 2min.Discard filtrate, again
Fill it up with supernatant, room temperature 2500g is centrifuged 3min.Repeat until having filtered all supernatants.
15th, add in 10mL C5 to Spin Filter, room temperature 2500g is centrifuged 3min.Discard filtrate.
16th, room temperature 2500g centrifugation 5min.
17th, in careful transfer Spin Filter to 2mL collecting pipe, avoid C5 contaminated aqueous solution as far as possible.
18th, add 5mL C6 solution to white filter membrane center.Room temperature 2500g is centrifuged 3min.
19th, discard Spin Filter.Now the DNA in collecting pipe can be directly used for downstream experiment, need not be pure further
Change.
Embodiment 1, comparative example 1, the extraction effect of comparative example 2 compare and are shown in Table 1.With regard to extraction time：Compared with lifting manipulation,
The extraction time of the present invention shortens nearly 1 half, is compared with traditional kit, and the time also shortens 0.5-1h；With regard to being extracted into
This：Compare with conventional reagents box, the extraction cost of the present invention reduces 6 times；With regard to extracting concentration：The concentration of the present invention is about hand
About the twice of formulation, suitable with the concentration of RNA isolation kit；With regard to purity：OD260 is the top that nucleic acid absorbs, and OD280 is
The top of absorbing proteins, OD230 is the absworption peak of organic reagent and polysaccharide, when OD260/280 is in 1.8-2.0, nucleic acid
Purity higher, pure nucleic acid OD260/230 should be more than or equal to 2.0, and the present invention and RNA isolation kit all can obtain purer
DNA, and lifting manipulation OD260/280 be less than 1.8, show to be vulnerable to the pollution of albumen, OD260/230 be less than 2.0, represent that this carries
Method is taken to be difficult to remove carbohydrate, polypeptide, phenol etc..To sum up, the present invention, on the premise of keeping high concentration and purity, shows
The cost and the time that reduce the extraction of fermented grain sample DNA writing, it is advantageous to the base that enterprise extracts fermented grain sample on a large scale
Because organizing DNA.
Table 1 embodiment 1, comparative example 1, the extraction effect of comparative example 2 compare
The different grinding agent effectiveness comparison of embodiment 4
1. respectively by the quartz sand of 5,12,16,20 and 25 mesh, and after 12 mesh, 16 mesh, 20 mesh bead sterilizing, dryings,
Weigh 18.5g to load in 50mL sterile centrifugation tube.
2. weigh 7.5g fermented grain sample in centrifuge tube, add 20mLCTAB extract, homogeneous instrument shakes 1min, 10000rpm
Centrifugation 10min.
3. shift supernatant to 50mL sterile centrifugation tube, plus equal-volume P:C:I (phenol：Chloroform：Isoamyl alcohol=25:24:
1), shake up, 11000rpm is centrifuged 20min.
4. shift supernatant to 50mL sterile centrifugation tube, plus 2 times of volume silica gel film combination liquid, shake up；Use 15mL adsorption column
Filter, abandon filtrate, till having filtered.
5. add 3.5mL 70% ethanol in adsorption column, filter, abandon filtrate.
6. repeat step 5 is once.
7.10000rpm blank pipe is centrifuged 2min, abandons filtrate.
8. 40min is dried under adsorption column natural conditions, adds 300 μ L sterilized water dissolving DNAs, filter, filtrate is loaded on 1.5mL
In sterile centrifugation tube, -20 DEG C save backup.
Extraction effect compares and is shown in Table 2.Although quartz sand flintiness is high, have edges and corners, and, someone discloses and uses stone
Sand extracts the DNA in fermented grain sample, can get good shell-broken effect.However, the quartz sand of not all particle diameter is equal
Extraction step that can be follow-up with the present invention matches, and extracts the high genomic DNA of amount of fine quality.Through the invention demonstrates that, less
Particle diameter (5 mesh) and larger particle diameter (25 mesh), its shell-broken effect is well below bead, the particle diameter only in particular range
When (12-16 mesh), its shell-broken effect of competence exertion, obtain the genomic DNA of high concentration (being shown in Table 2) high-quality (being shown in Table 1).
The extraction effect of the grinding agent of table 2 different-grain diameter compares
Claims (10)
1. in a kind of economic rapid extraction fermented grain microbe genome DNA method, comprise the following steps：
1) microbial cell broken wall in fermented grain：Shake at a high speed after grinding agent, fermented grain sample and lysate are mixed, be then centrifuged for；
Described grinding agent is quartz sand；Further, described quartz sand particle size is 12-20 mesh；
2) nucleic acid purification.
2. the method for claim 1 is it is characterised in that described quartz sand particle size is 16 mesh.
3. the method described in claim 1 is it is characterised in that step 2) nucleic acid purification comprise the steps of：
A) removal of impurities：Take step 1) in centrifugation after supernatant add P.C.I mix, centrifugation；
B) cross post：Supernatant after centrifugation is combined liquid with pellosil mix, then crosses nucleic acid purification post, filtrate is abandoned in centrifugation；Institute
The pellosil stated combines the mixed liquor that liquid is that pH is the guanidine hydrochloride of 4-6.5 and potassium acetate；
C) wash post：Add eluent to cross nucleic acid purification post, then high speed centrifugation dries nucleic acid purification post, be dried under natural conditions；
D) elute：Eluent dissolving DNA, the lower DNA solution of centrifugation wash-out are added on the pellosil of nucleic acid extraction column.
4. the method for claim 1 is it is characterised in that step 1) in,
The ratio of grinding agent, fermented grain sample and lysate three is followed successively by 15～22g:6～9g:16～24mL；Preferably, it is
18.5g:7.5g:20mL.
5. the method for claim 1 is it is characterised in that step 1) in,
Cracking formula of liquid is as follows：1.5-2.5%CTAB (w/v), 90-112mM Tris-HCl (pH 8.0), 18-24mM EDTA
(pH8.0), 1.2-1.6M NaCl；Preferably 2%CTAB (w/v), 100mM Tris-HCl (pH 8.0), 20mM EDTA
(pH8.0), 1.4M NaCl.
6. method as claimed in claim 3 is it is characterised in that in step a),
Described P.C.I is 24-26 volume of phenol：23-25 volume of chloroform：0.5-1.5 volume isoamyl alcohol；Preferably 25 volume benzene
Phenol：24 volume of chloroform：1 volume isoamyl alcohol；
P.C.I is 1 with the volume ratio of supernatant:0.8～1.2.
7. method as claimed in claim 3 is it is characterised in that in step b),
The volume ratio that supernatant is combined liquid with pellosil is 1:1.5～2.5；
The mixed liquor of guanidine hydrochloride and potassium acetate that pellosil is 5 with reference to liquid for pH；
Guanidine hydrochloride with the molal weight ratio of potassium acetate is：3-4M：0.5M；Preferably 4M：0.5M.
8. the method as described in any one of right 1-7 is it is characterised in that comprise the following steps：
After the quartz sand sterilizing, drying of 12～20 mesh, weigh 15～22g and load in 50mL sterile centrifugation tube；
Weigh 6～9g fermented grain sample in centrifuge tube, add 16～24mL lysate, homogeneous instrument shakes 1min, 10000rpm is centrifuged
10min；
Transfer supernatant, to 50mL sterile centrifugation tube, plus 0.8～P of 1.2mL:C:I, shakes up, and 11000rpm is centrifuged 20min；Institute
The P.C.I stating is 25 volume of phenol：24 volume of chloroform：1 volume isoamyl alcohol；
Transfer supernatant, to 50mL sterile centrifugation tube, plus 1.5～pellosil of 2.5mL combines liquid, shakes up；Use 15mL nucleic acid purification
Post filters, and abandons filtrate, till having filtered；
Jia 2.5 in adsorption column～3.5mL 70% ethanol, filter, abandon filtrate, be repeated once；
10000rpm blank pipe is centrifuged 2min, abandons filtrate；
30～60min is dried under purification column natural conditions, adds 50～300 μ L aseptic deionized waters or TE solution dissolving DNA, mistake
Filter, filtrate is loaded in 1.5mL sterile centrifugation tube, and -20 DEG C save backup.
9. method as claimed in claim 8 is it is characterised in that described cracking formula of liquid is 2%CTAB (w/v), 100mM
Tris-HCl (pH 8.0), 20mM EDTA (pH8.0), 1.4M NaCl.
10. method as claimed in claim 8 is it is characterised in that it is 5 that described pellosil combines liquid pH；Described pellosil
It is 4M guanidine hydrochloride in conjunction with liquid：0.5M potassium acetate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610806782.7A CN106399297B (en) | 2016-09-07 | 2016-09-07 | Method for economically and rapidly extracting microbial genome DNA (deoxyribonucleic acid) in fermented grains |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610806782.7A CN106399297B (en) | 2016-09-07 | 2016-09-07 | Method for economically and rapidly extracting microbial genome DNA (deoxyribonucleic acid) in fermented grains |
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