CN106754888A - The extracting method and kit of a kind of grand genome DNA of fish enteric microorganism - Google Patents
The extracting method and kit of a kind of grand genome DNA of fish enteric microorganism Download PDFInfo
- Publication number
- CN106754888A CN106754888A CN201710047905.8A CN201710047905A CN106754888A CN 106754888 A CN106754888 A CN 106754888A CN 201710047905 A CN201710047905 A CN 201710047905A CN 106754888 A CN106754888 A CN 106754888A
- Authority
- CN
- China
- Prior art keywords
- solution
- fish
- dna
- tris
- gel
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The extracting method and kit of a kind of grand genome DNA of fish enteric microorganism that the present invention is provided, it includes：(1) fish sample sampling and fish superficial microbes fixing process；(2) acquisition of enteric microorganism and the collection of thalline；(3) embedding of cell and cracking；(4) extraction of DNA；(5) DNA is preserved.Kit main component：Solution A：It is the mixed solution of Tris, EDTA；B is Tris, EDTA, NaCl, PVP, guanidinium isothiocyanate, the mixed solution of Triton X100, PMSF, β mercaptoethanols；C is the mixed solution of Tris, EDTA, NaCl and DTT；Solution D is Proteinase K, lysozyme, the mixed solution of Tris, NaCl, SDS；E is Tris saturated phenols and chloroform mixed liquor；F is isopropanol；G is ethanol water.The DNA fragmentation purity that the present invention is obtained is high, and host and host body surface microbial DNA content are few.
Description
Technical field
Field, more particularly to a kind of fish enteric microorganism Metagenomic library construction STb gene are extracted the present invention relates to DAN
Extracting method and kit.
Background technology
Microbe research means traditional at present can only cultivate 0.1%~1% environmental microorganism, and this will be unable to overall point
Analysis and the composition and function using microbiologic population.Grand genomic library technology can avoid the limitation to microorganism pure culture,
DNA molecular is directly obtained from environment, by sequencing or library screening, for the mankind provide further with microbial resources
Important means.The extraction and purification of DNA is the first step of Metagenomic library construction, is also a step of most critical.
Fish enteric microorganism has very important researching value, can be used for researching fish health and Nutrition and Metabolism, obtains
Obtain new enzyme preparation etc..Conventional DNA is extracted referring especially to faeces DNA extraction method.But due to being obtained in library construction process
High-purity and high-quality DNA, should try one's best and avoid the pollution of fish superficial microbes, fish intestines tissue and food, while also
Avoid degraded of the fish digestive tube digestive juice to DNA.Thus, it is grand that conventional DNA extraction method can not meet fish enteric microorganism
The requirement of genomic library construction.So, prior art needs further improvement and develops.
The content of the invention
In view of above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of grand genome of fish enteric microorganism
The extracting method and kit of STb gene, on the premise of cost-effective, extract high-purity DNA.
In order to solve the above technical problems, the present invention program includes：
A kind of kit for the grand genome DNA extracting method of fish enteric microorganism, it includes：
Solution A：100mM/L Tris-HCl, 1mM/L EDTA, pH 8.0；
Solution B：100mM/L Tris-HCl, 20mM/L EDTA, 100mM/L NaCl, 0.1g/L PVP, 100mM/L are different
Guanidine thiocyanate, 0.1%Triton-X100,0.1mM PMSF, 1% (v/v) beta -mercaptoethanol, pH 8.5；
Solution C：100mM/L Tris-HCl, 1mM/L EDTA, 0.9%NaCl, 5mM/L DTT, pH 8.0；
Solution D：50mg/ml Proteinase Ks, 20mg/ml lysozymes, 100mM/L Tris-HCl, 100mM/L NaCl,
0.1%SDS, pH 8.0；
Solution E：50%Tris-HCl saturated phenols (pH 8.0), 48% chloroform, 2% isoamyl alcohol；
Solution F：96% chloroform, 4% isoamyl alcohol；
Solution G：Isopropanol；
Solution H：70% ethanol.
Using the extracting method of the grand genome DNA of fish enteric microorganism of the kit, it is comprised the following steps：
Step one, fresh Fish Sample is hit unconsciously, Fish Sample body surface liquid is blotted with blotting paper, with 75% alcohol wipe
Fish Sample body surface face, being placed in -20 DEG C of refrigerators carries out freezing 15-30 minutes；
Step 2, Ago-Gel solution is prepared using solution A, it is equilibrated to 65 DEG C in incubator；
Step 3 is rapid to take out the Fish Sample that above-mentioned steps one are obtained, the Ago-Gel solution obtained using step 2
Fish Sample superficial microbes are fixed by coated Fish Sample body surface；Fish body is carried out rapidly using sterilizing operating scissors after cooling
Dissect, isolated fish enteron aisle；
Step 4, the solution B for drawing corresponding amount using sterilizing filter is slowly injected into the fish enteron aisle that above-mentioned steps three are obtained,
Repeat wash-out 3 times, the fish intestinal contents collection for obtaining will be rinsed and obtain fish intestinal contents solution；
Step 5, the fish intestinal contents solution that above-mentioned steps four are obtained is placed in triangular flask, adds bead whirlpool
Concussion 10-15 minutes, obtains suspension；
Step 6,8 layer of 80 mesh nylon grenadine is placed on Buchner funnel, and the suspension that suction filtration above-mentioned steps five are obtained makes
Sediment is cleaned with the solution B of corresponding amount, and collects filtrate；
Step 7, the filtrate obtained by step 6 is centrifuged 10 minutes under the conditions of 10000rpm and collected precipitation, Ran Houyong
The resuspended precipitation of solution C, is repeated 3 times, and obtains hypothallus precipitation standby；
Step 8, using the low melting-point agarose gel that solution A configuration concentration is 1%, balances its temperature to 45 DEG C；Will step
After rapid seven bacterial sediments for obtaining are preheating to 45 DEG C, the 1% low melting-point agarose gel is added thereto to, is noted after being well mixed
Enter mould, and solidified 12 hours at 4 DEG C, obtain gel；
Step 9, in the gel immersion solution D that step 8 is obtained, isothermal vibration is cleaned 12 hours at 37 DEG C；
Step 10, the gel after cleaning is put into low melting-point agarose electrophoresis tank, is coagulated using 1% low melting-point agarose
Glue is closed, electrophoresis 10 minutes under the conditions of 60v, and cuts agar rapidly under uviol lamp, obtains Ago-Gel；
Step 11, after the Ago-Gel that step 10 is cut adds isometric solution A, is heated to 65 DEG C of thawings,
And isometric solution E is added immediately, and mixing is rocked, it is centrifuged 5 minutes under the conditions of 12000rpm, obtain water phase；
Step 12, the water that step 11 is obtained mutually is moved on in another 1.5mL Eppendorf pipes, adds same volume
Solution F, under the conditions of 12000rpm be centrifuged 5 minutes, obtain corresponding water phase,
Step 13, during the corresponding water that step 12 is obtained mutually moved on into another 1.5mL Eppendorf pipes, adds 2 times
Volume cooled solution G, staticly settles 12 hours under the conditions of -80 DEG C, is then centrifuged 10 minutes under the conditions of 10000rpm is carried out；
Then it is added thereto to a small amount of Solution H repeated washing 2 times, obtains DNA precipitations；
Step 14, the DNA precipitations that step 13 is obtained are dried at room temperature, and removal alcohol is to experimental result
Influence, the solution A dissolving for being subsequently adding respective amount obtains corresponding DNA.
The extracting method and kit of a kind of grand genome DNA of fish enteric microorganism that the present invention is provided, are prevented effectively from
Host, host body surface microorganism, the pollution of food texture DNA, the DNA fragmentation for obtaining do not have RNA, protein, polysaccharide, phenols
Deng the pollution of material, the treatment of later experiments is simplified, the integrality of DNA molecular chain is obviously improved than conventional method.Obtained
DNA can meet Metagenomic library construction, grand genomic library sequencing, PCR analysis etc. requirement of experiment.
Brief description of the drawings
Fig. 1 is the electrophoretogram of the little yellow croaker enteric microorganism STb gene of extraction in the present invention；
Fig. 2 is the electrophoretogram for carrying out 16S-rRNA gene PCR amplifications in the present invention using kit.
Specific embodiment
The invention provides the extracting method and kit of a kind of grand genome DNA of fish enteric microorganism, to send out this
Bright purpose, technical scheme and effect are clearer, clear and definite, and the present invention is described in more detail below.It should be appreciated that herein
Described specific embodiment is only used to explain the present invention, is not intended to limit the present invention.
The invention provides a kind of kit for the grand genome DNA extracting method of fish enteric microorganism, it includes：
Solution A：100mM/L Tris-HCl, 1mM/L EDTA, pH 8.0；
Solution B：100mM/L Tris-HCl, 20mM/L EDTA, 100mM/L NaCl, 0.1g/L PVP, 100mM/L are different
Guanidine thiocyanate, 0.1% (v/v) Triton-X100,0.1mM PMSF, 1% (v/v) beta -mercaptoethanol, pH 8.5；
Solution C：100mM/L Tris-HCl, 1mM/L EDTA, 0.9%NaCl, 5mM/L DTT, pH 8.0；
Solution D：50mg/ml Proteinase Ks, 20mg/ml lysozymes, 100mM/L Tris-HCl, 100mM/L NaCl,
0.1%SDS, pH 8.0；
Solution E：50%Tris-HCl saturated phenols (pH 8.0), 48% chloroform, 2% isoamyl alcohol；
Solution F：96% chloroform, 4% isoamyl alcohol；
Solution G：Isopropanol；
Solution H：70% ethanol.
It is more specifically：
Solution A is the mixed solution of Tris, EDTA, and Tris concentration is 100mM/L in mixed solution, and EDTA concentration is 1mM/
L, pH to 8.0 is adjusted with hydrochloric acid.
Solution B is Tris, EDTA, NaCl, PVP, guanidinium isothiocyanate, PMSF, the mixed solution of beta -mercaptoethanol, is mixed molten
Tris concentration is 100mM/L in liquid, and EDTA concentration is 20mM/L, and NaCl concentration is 100mM/L, and PVP concentration is 0.1g/L, different sulphur
Cyanic acid guanidine concentration is 100mM/L, and PMSF concentration is 100mM/L, and Triton-X100 concentration is 0.1% (v/v), beta -mercaptoethanol
Concentration is 1% (v/v), and pH to 8.5 is adjusted with hydrochloric acid.
Solution C is the mixed solution of Tris, EDTA, NaCl.Tris concentration is 100mM/L, EDTA concentration in mixed solution
It is 1mM/L, NaCl concentration is that 0.9%, DTT concentration is 5mM/L, and pH to 8.0 is adjusted with hydrochloric acid.
Solution D is Proteinase K, lysozyme, the mixed solution of Tris, NaCl, SDS, and wherein proteinase K concentration is 50mg/
ML, lysozyme concentration is 20mg/mL, and Tris100mM/L, NaCl concentration is 100mM/L, and SDS concentration is 0.1%, is adjusted with hydrochloric acid
Whole pH to 8.0.
Solution E is Tris saturated phenols (pH 8.0), chloroform and isoamyl mixed alkoxide solution.Tris saturated phenols (pH 8.0) volume
Fraction is 50%, and chloroform volume fraction is 48%, and isoamyl alcohol volume fraction is 2%.
Solution F is chloroform isoamyl alcohol mixed solution, and chloroform volume fraction is 96%, and isoamyl alcohol volume fraction is 4%.Solution
G is isopropanol, and Solution H is 70% ethanol water.
Its specific effect is as follows：Solution A is used to configure Ago-Gel solution, and in such circumstances, microbial DNA can be with
Keep stabilization.Solution B is used for the flushing and collection of fish intestinal contents, and the reagent can inactivate or suppress DNA enzymatic, protease etc.
The activity of enzyme, reduces the degraded of microorganism in content.Solution C causes enteric microorganism cell to crack and DNA for dilution
The enzyme and its inhibitor of hydrolysis, stabilization enteric microorganism cell.Solution D is used to crack the microbial cell in blob of viscose.
Solution E is denatured the protein in the blob of viscose of thawing and it is separated with DNA, the impurity such as extraction part sugar, lipid.Solution
F makes residual Tris phenol be dissolved into organic phase, further goes the removal of impurity.The effect of solution G is precipitation DNA.The effect of Solution H is
Cleaning and precipitation DNA.
Present invention also offers a kind of extraction side of the grand genome DNA of fish enteric microorganism of use mentioned reagent box
Method, it is comprised the following steps：
Step one, fresh Fish Sample is hit unconsciously, Fish Sample body surface liquid is blotted with blotting paper, with 75% alcohol wipe
Fish Sample body surface face, being placed in -20 DEG C of refrigerators carries out freezing 15-30 minutes；
Step 2, Ago-Gel solution is prepared using solution A, it is equilibrated to 65 DEG C in incubator；
Step 3 is rapid to take out the Fish Sample that above-mentioned steps one are obtained, the Ago-Gel solution obtained using step 2
Fish Sample superficial microbes are fixed by coated Fish Sample body surface；Fish body is carried out rapidly using sterilizing operating scissors after cooling
Dissect, isolated fish enteron aisle；
Step 4, the solution B for drawing corresponding amount using sterilizing filter is slowly injected into the fish enteron aisle that above-mentioned steps three are obtained,
Repeat wash-out 3 times, the fish intestinal contents collection for obtaining will be rinsed and obtain fish intestinal contents solution；
Step 5, the fish intestinal contents solution that above-mentioned steps four are obtained is placed in triangular flask, adds bead whirlpool
Concussion 10-15 minutes, obtains suspension；
Step 6,8 layer of 80 mesh nylon grenadine is placed on Buchner funnel, and the suspension that suction filtration above-mentioned steps five are obtained makes
Sediment is cleaned with the solution B of corresponding amount, and collects filtrate；
Step 7, the filtrate obtained by step 6 is centrifuged 10 minutes under the conditions of 10000rpm and collected precipitation, Ran Houyong
The resuspended precipitation of solution C, is repeated 3 times, and obtains hypothallus precipitation standby；
Step 8, using the low melting-point agarose gel that solution A configuration concentration is 1%, balances its temperature to 45 DEG C；Will step
After rapid seven bacterial sediments for obtaining are preheating to 45 DEG C, the 1% low melting-point agarose gel is added thereto to, is noted after being well mixed
Enter mould, and solidified 12 hours at 4 DEG C, obtain gel；
Step 9, in the gel immersion solution D that step (8) is obtained, isothermal vibration is cleaned 12 hours at 37 DEG C；
Step 10, the gel after cleaning is put into low melting-point agarose electrophoresis tank, is coagulated using 1% low melting-point agarose
Glue is closed, electrophoresis 10 minutes under the conditions of 60v, and cuts agar rapidly under uviol lamp, obtains Ago-Gel；
Step 11, after the Ago-Gel that step 10 is cut adds isometric solution A, is heated to 65 DEG C of thawings,
And isometric solution E is added immediately, and mixing is rocked, it is centrifuged 5 minutes under the conditions of 12000rpm, obtain water phase；
Step 12, the water that step 11 is obtained mutually is moved on in another 1.5mL Eppendorf pipes, adds same volume
Solution F, under the conditions of 12000rpm be centrifuged 5 minutes, obtain corresponding water phase,
Step 13, during the corresponding water that step 12 is obtained mutually moved on into another 1.5mL Eppendorf pipes, adds 2 times
Volume cooled solution G, staticly settles 12 hours under the conditions of -80 DEG C, is then centrifuged 10 minutes under the conditions of 10000rpm is carried out；
Then it is added thereto to a small amount of Solution H repeated washing 2 times, obtains DNA precipitations；
Step 14, the DNA precipitations that step 13 is obtained are dried at room temperature, and removal alcohol is to experimental result
Influence, the solution A dissolving for being subsequently adding respective amount obtains corresponding DNA
In order to more description is of the invention in detail, it is exemplified below more detailed embodiment and illustrates.
(1) fresh little yellow croaker is hit unconsciously, body surface liquid is blotted with blotting paper, with 75% alcohol wipe fish body surface,
Being placed in -20 DEG C of refrigerators carries out freezing 15-30 minutes；
(2) Ago-Gel solution is prepared using solution A, it is equilibrated to 65 DEG C in incubator；
(3) fish body that above-mentioned steps (1) are obtained is taken out rapidly, and the Ago-Gel solution obtained using step (2) is coated
Its superficial microbes is fixed by fish body table, fish body is dissected rapidly using sterilizing operating scissors after cooling, careful separation
Obtain fish enteron aisle；
(4) solution B for drawing 5mL using sterilizing filter is slowly injected into the fish enteron aisle that above-mentioned steps (3) are obtained, and repetition is washed
It is de- 3 times, the fish intestinal contents collection for obtaining will be rinsed and obtain fish intestinal contents solution；
(5) the fish intestinal contents solution for obtaining above-mentioned steps (4) is placed in triangular flask, adds the concussion of bead whirlpool
10-15 minutes, obtain suspension；
(6) 8 layer of 80 mesh nylon grenadine is placed on Buchner funnel, the suspension that suction filtration above-mentioned steps (5) are obtained is used
The solution B cleaning sediment of 15mL, and collect filtrate；
(7) filtrate obtained by step (6) is centrifuged 10 minutes under the conditions of 10000rpm and collects precipitation, then use solution
The resuspended precipitations of C, are repeated 3 times, and obtain hypothallus precipitation standby；
(8) using the low melting-point agarose gel that solution A configuration concentration is 1%, its temperature to 45 DEG C is balanced；By step
(7) after the bacterial sediment for obtaining is preheating to 45 DEG C, the 1% low melting-point agarose gel is added thereto to, is noted after being well mixed
Enter mould, and solidified 12 hours at 4 DEG C, obtain gel；
(9) in the gel immersion solution D for obtaining step (8), isothermal vibration is cleaned 12 hours at 37 DEG C；
(10) gel after cleaning is put into low melting-point agarose electrophoresis tank, uses 1% low melting-point agarose gel
Closed, electrophoresis 10 minutes under the conditions of 60v, and agar is cut rapidly under uviol lamp, obtained Ago-Gel；
(11) after the Ago-Gel for cutting step (10) adds isometric solution A, 65 DEG C of thawings are heated to, are existed side by side
Isometric solution E is added, mixing is rocked, is centrifuged 5 minutes under the conditions of 12000rpm, obtain water phase；
(12) in the water that step (11) is obtained mutually being moved on into another 1.5mL Eppendorf pipes, the molten of same volume is added
Liquid F, is centrifuged 5 minutes under the conditions of 12000rpm, obtains corresponding water phase；
(13) in the corresponding water that step (12) is obtained mutually being moved on into another 1.5mL Eppendorf pipes, add 2 times of volumes pre-
Cold soln G；
(14) staticly settled under the conditions of -80 DEG C 12 hours, be then centrifuged 10 minutes under the conditions of 10000rpm is carried out；
(15) and then it is added thereto to a small amount of Solution H repeated washing 2 times, obtains DNA precipitations；
(16) the DNA precipitations for obtaining step (15) are dried at room temperature, influence of the removal alcohol to experimental result；
(17) the solution A dissolving of 100 μ L is added to obtain corresponding DNA.
As shown in Fig. 1 and table 1, table 1 is to use nanodrop ultramicron scene luminometric analysis sample concentration and pure
Degree result.
Table 1
Wherein, sample 1：For kit of the present invention extracts sample；M：Molecular weight mark marker；Sample 2：For SDS methods are extracted
Sample；Sample 3：For CTAB methods extract sample；Sample 4：For column chromatography kit 1 extracts sample；Sample 5：It is column chromatography reagent
Box 2 extracts sample；Sample 6：For column chromatography kit 3 extracts sample.Show：Extract what is obtained using conventional SDS and CTAB methods
DNA there occurs serious degraded, it is impossible to reach the requirement of grand genome research；Obtained using column chromatography DNA extraction kit method
DNA sample band disperse, also there is a certain degree of degraded and protein contamination.The DNA sample band extracted using this kit is clear
Clear, degradation amount is few, does not have protein contamination and RNA to remain, and the purity and quality of the DNA for being obtained are high.
It is more specifically as shown in Figure 2, wherein, sample 7 is blank, and sample 8 is to be increased with little yellow croaker enteron aisle DNA sample
The 16s rRNA genes for obtaining, sample 9 is the 16s rRNA genes obtained with the amplification of snakeheaded fish enteron aisle DNA sample；M：Molecular weight mark
marker.From figure 2 it can be seen that can be amplified with the soil DNA bright, it is contemplated that the single-minded purpose band of size, table
The bright fish enteron aisle DNA extracted using the DNA extraction method and kit is met regular-PCR and expands requirement.Use the DNA extraction sides
Method and kit extract snakeheaded fish and little yellow croaker intestinal microbial DNA, and 5.11G is obtained respectively by illumina high-flux sequences
With the sequencing data of 5.20G, found after being analyzed using SoapAligner softwares, host and scratch sufficient class biology DNA and only account for survey
The 0.01% of ordinal number evidence and 0.03%.Show the extracting method it is possible to prevente effectively from fish body in itself with the pollution of food DNA.
Certainly, described above is only presently preferred embodiments of the present invention, and the present invention is not limited to enumerate above-described embodiment, should
When explanation, any those of ordinary skill in the art are all equivalent substitutes for being made, bright under the teaching of this specification
Aobvious variant, all falls within the essential scope of this specification, ought to be subject to protection of the invention.
Claims (2)
1. a kind of kit for the grand genome DNA extracting method of fish enteric microorganism, it includes：
Solution A：100mM/L Tris-HCl, 1mM/L EDTA, pH 8.0；
Solution B：100mM/L Tris-HCl, 20mM/L EDTA, 100mM/L NaCl, the 0.1g/L different sulphur cyanogen of PVP, 100mM/L
Sour guanidine, 0.1%Triton-X100,0.1mM PMSF, 1% (v/v) beta -mercaptoethanol, pH 8.5；
Solution C：100mM/L Tris-HCl, 1mM/L EDTA, 0.9%NaCl, 5mM/L DTT, pH 8.0；
Solution D：50mg/ml Proteinase Ks, 20mg/ml lysozymes, 100mM/L Tris-HCl, 100mM/L NaCl, 0.1%
SDS, pH 8.0；
Solution E：50%Tris-HCl saturated phenols (pH 8.0), 48% chloroform, 2% isoamyl alcohol；
Solution F：96% chloroform, 4% isoamyl alcohol；
Solution G：Isopropanol；
Solution H：70% ethanol.
2. a kind of extracting method of grand genome DNA of fish enteric microorganism using kit as claimed in claim 1, its bag
Include following steps：
Step one, fresh Fish Sample is hit unconsciously, Fish Sample body surface liquid is blotted with blotting paper, with 75% alcohol wipe sample
Fish body surface, being placed in -20 DEG C of refrigerators carries out freezing 15-30 minutes；
Step 2, Ago-Gel solution is prepared using solution A, it is equilibrated to 65 DEG C in incubator；
Step 3, rapid to take out the Fish Sample that above-mentioned steps one are obtained, the Ago-Gel solution obtained using step 2 is coated
Fish Sample superficial microbes are fixed by Fish Sample body surface；Fish body is dissected rapidly using sterilizing operating scissors after cooling,
Isolated fish enteron aisle；
Step 4, the solution B for drawing corresponding amount using sterilizing filter is slowly injected into the fish enteron aisle that above-mentioned steps three are obtained, and repeats
Wash-out 3 times, will rinse the fish intestinal contents collection for obtaining and obtain fish intestinal contents solution；
Step 5, the fish intestinal contents solution that above-mentioned steps four are obtained is placed in triangular flask, adds the concussion of bead whirlpool
10-15 minutes, obtain suspension；
Step 6,8 layer of 80 mesh nylon grenadine is placed on Buchner funnel, the suspension that suction filtration above-mentioned steps five are obtained, using right
The solution B cleaning sediment that should be measured, and collect filtrate；
Step 7, the filtrate obtained by step 6 is centrifuged 10 minutes under the conditions of 10000rpm and precipitation is collected, and then uses solution C
Resuspended precipitation, is repeated 3 times, and obtains hypothallus precipitation standby；
Step 8, using the low melting-point agarose gel that solution A configuration concentration is 1%, balances its temperature to 45 DEG C；By step 7
After the bacterial sediment for obtaining is preheating to 45 DEG C, the 1% low melting-point agarose gel is added thereto to, mould is injected after being well mixed
Tool, and solidified 12 hours at 4 DEG C, obtain gel；
Step 9, in the gel immersion solution D that step 8 is obtained, isothermal vibration is cleaned 12 hours at 37 DEG C；
Step 10, the gel after cleaning is put into low melting-point agarose electrophoresis tank, and the low melting-point agarose gel using 1% enters
Row closing, electrophoresis 10 minutes under the conditions of 60v, and agar is cut rapidly under uviol lamp, obtain Ago-Gel；
Step 11, after the Ago-Gel that step 10 is cut adds isometric solution A, is heated to 65 DEG C of thawings, exists side by side
Isometric solution E is added, mixing is rocked, is centrifuged 5 minutes under the conditions of 12000rpm, obtain water phase；
Step 12, the water that step 11 is obtained mutually is moved on in another 1.5mL Eppendorf pipes, adds the molten of same volume
Liquid F, is centrifuged 5 minutes under the conditions of 12000rpm, obtains corresponding water phase,
Step 13, during the corresponding water that step 12 is obtained mutually moved on into another 1.5mL Eppendorf pipes, adds 2 times of volumes
Cooled solution G, staticly settles 12 hours under the conditions of -80 DEG C, is then centrifuged 10 minutes under the conditions of 10000rpm is carried out；Then
It is added thereto to a small amount of Solution H repeated washing 2 times, obtains DNA precipitations；
Step 14, the DNA precipitations that step 13 is obtained are dried at room temperature, shadow of the removal alcohol to experimental result
Ring, the solution A dissolving for being subsequently adding respective amount obtains corresponding DNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710047905.8A CN106754888B (en) | 2017-01-21 | 2017-01-21 | A kind of extracting method and kit of the macro genome DNA of fish enteric microorganism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710047905.8A CN106754888B (en) | 2017-01-21 | 2017-01-21 | A kind of extracting method and kit of the macro genome DNA of fish enteric microorganism |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106754888A true CN106754888A (en) | 2017-05-31 |
| CN106754888B CN106754888B (en) | 2019-11-22 |
Family
ID=58941451
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710047905.8A Active CN106754888B (en) | 2017-01-21 | 2017-01-21 | A kind of extracting method and kit of the macro genome DNA of fish enteric microorganism |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106754888B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107326063A (en) * | 2017-07-31 | 2017-11-07 | 农业部沼气科学研究所 | A kind of method that fixation of bacteria observation is counted |
| CN107475250A (en) * | 2017-09-15 | 2017-12-15 | 广东美格基因科技有限公司 | A kind of pre-treating method of intestinal contents macro genome DNA extraction |
| CN108048455A (en) * | 2018-02-10 | 2018-05-18 | 中国水产科学研究院黄海水产研究所 | A kind of fish superficial microbes genome DNA extracting method |
| CN109609496A (en) * | 2019-01-16 | 2019-04-12 | 浙江工商大学 | A kind of extracting method of the feces of livestock and poultry total DNA for PCR amplification |
| CN109825499A (en) * | 2019-04-18 | 2019-05-31 | 华南农业大学 | A kind of extraction reagent, kit and the application and extracting method of cell-bacterial parasite total DNA |
| CN110904093A (en) * | 2019-11-29 | 2020-03-24 | 浙江万里学院 | Extraction method of fish intestinal bacterial community genome DNA |
| CN111454941A (en) * | 2020-04-13 | 2020-07-28 | 武汉轻工大学 | Sampling liquid and sampling method of DNA viruses in aerosol |
| CN114276932A (en) * | 2022-02-12 | 2022-04-05 | 合肥巅峰生物科技有限公司 | Microbial cell lysate |
| CN116949036A (en) * | 2023-09-11 | 2023-10-27 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from ascites and extraction method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103103180A (en) * | 2011-11-11 | 2013-05-15 | 新疆师范大学 | Method for separation and purification of large-fragment DNA from soil |
| CN105483120A (en) * | 2016-02-16 | 2016-04-13 | 天津农学院 | High-temperature combined method for extracting genomic DNA of fish enteric microorganisms |
-
2017
- 2017-01-21 CN CN201710047905.8A patent/CN106754888B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103103180A (en) * | 2011-11-11 | 2013-05-15 | 新疆师范大学 | Method for separation and purification of large-fragment DNA from soil |
| CN105483120A (en) * | 2016-02-16 | 2016-04-13 | 天津农学院 | High-temperature combined method for extracting genomic DNA of fish enteric microorganisms |
Non-Patent Citations (2)
| Title |
|---|
| 汪立平等: "罗非鱼幼鱼肠道微生物基因组DNA的提取", 《湖南农业科学》 * |
| 许尤厚等: "几种近江牡蛎肠道微生物DNA提取方法的比较", 《钦州学院学报》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107326063A (en) * | 2017-07-31 | 2017-11-07 | 农业部沼气科学研究所 | A kind of method that fixation of bacteria observation is counted |
| CN107475250A (en) * | 2017-09-15 | 2017-12-15 | 广东美格基因科技有限公司 | A kind of pre-treating method of intestinal contents macro genome DNA extraction |
| CN107475250B (en) * | 2017-09-15 | 2019-04-05 | 广东美格基因科技有限公司 | A kind of pre-treating method that intestinal contents macro genome DNA extracts |
| CN108048455A (en) * | 2018-02-10 | 2018-05-18 | 中国水产科学研究院黄海水产研究所 | A kind of fish superficial microbes genome DNA extracting method |
| CN109609496A (en) * | 2019-01-16 | 2019-04-12 | 浙江工商大学 | A kind of extracting method of the feces of livestock and poultry total DNA for PCR amplification |
| CN109825499A (en) * | 2019-04-18 | 2019-05-31 | 华南农业大学 | A kind of extraction reagent, kit and the application and extracting method of cell-bacterial parasite total DNA |
| CN109825499B (en) * | 2019-04-18 | 2020-11-06 | 华南农业大学 | Extraction reagent and kit for total DNA of cell-parasitic bacteria, application and extraction method |
| CN110904093A (en) * | 2019-11-29 | 2020-03-24 | 浙江万里学院 | Extraction method of fish intestinal bacterial community genome DNA |
| CN111454941A (en) * | 2020-04-13 | 2020-07-28 | 武汉轻工大学 | Sampling liquid and sampling method of DNA viruses in aerosol |
| CN114276932A (en) * | 2022-02-12 | 2022-04-05 | 合肥巅峰生物科技有限公司 | Microbial cell lysate |
| CN116949036A (en) * | 2023-09-11 | 2023-10-27 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from ascites and extraction method |
| CN116949036B (en) * | 2023-09-11 | 2023-12-15 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from ascites and extraction method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106754888B (en) | 2019-11-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754888B (en) | A kind of extracting method and kit of the macro genome DNA of fish enteric microorganism | |
| US7893251B2 (en) | Methods for selective isolation of nucleic acids from microbial cells present in samples containing higher eukaryotic cells and/or tissues | |
| EP1756136B1 (en) | Kits and processes for removing contaminants from nucleic acids in environmental and biological samples | |
| EP1735440B1 (en) | Reagents and methods for isolation of purified rna | |
| Siddhapura et al. | Comparative studies on the extraction of metagenomic DNA from the saline habitats of Coastal Gujarat and Sambhar Lake, Rajasthan (India) in prospect of molecular diversity and search for novel biocatalysts | |
| CN102586234B (en) | Method for extracting high-molecular-weight genome from animal feces | |
| KR101639650B1 (en) | Method for separating and purifying rna from biomaterial | |
| CN101712953B (en) | DNA extracting method for evaluating community diversity of the intestinal microorganisms of animals | |
| US20120171728A1 (en) | Process for amplifying dna using tetratethylene glycol, kit of parts therefor and use thereof | |
| CN101709298B (en) | Soil DNA extracting method for evaluating diversity of microbial community of plant root system | |
| JP2011045386A (en) | Method of collecting dna from environmental sample | |
| Jahan et al. | Genomic DNA extraction methods: a comparative case study with gram–negative organisms | |
| CN1978453A (en) | Method for extracting soil microbial DNA | |
| Moslem et al. | An efficient method for DNA extraction from Cladosporioid fungi | |
| CN112646806A (en) | Rapid extraction method and kit for soil DNA | |
| CN102643795A (en) | Method for extracting total DNAs of soil microorganisms through PVP pretreatment | |
| CN112501156A (en) | High-efficiency extraction method of total DNA of marine shellfish biological sediment | |
| CN114703231B (en) | Electroporation gene editing method and application of crassostrea gigas beta-tubulin gene | |
| Merante et al. | Isolation of total cellular DNA from tissues and cultured cells | |
| Bailes et al. | MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY | |
| Bahkali et al. | In-house protocol for isolation of restrictable and amplifiable genomic DNA from plants, fungi and bacteria | |
| CN117248032A (en) | Kit for detecting invasive species based on Crispr/Cas12a system and application thereof | |
| JP5277497B2 (en) | Purification of RNA extracted from soil | |
| KR20120053831A (en) | Primer set of polymerase chain reaction for detecting xanthomonas campestris pv. incanae and method for detecting xanthomonas campestris pv. incanae using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |