CN102978198A - Microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora - Google Patents
Microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora Download PDFInfo
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Abstract
The invention relates to a microbial genome DNA (deoxyribonucleic acid) indirect extraction method for evaluating diversity of animal intestinal microflora. Before cell lysis, the method performs pretreatment on a sample and separates microbial cells from the excrement sample to avoid the problems that pollutants are difficult to remove and the DNA recovery rate is low in a purification step. According to the invention, phenol and chloroform are not used in the extraction process, thus harm to the physical health of experimenters is reduced. The OD260/OD230 and OD260/OD280 of the extracted intestinal microorganism DNA are approximate to standard values, and the intestinal microorganism DNA can be directly applied to molecular operation to evaluate the diversity of animal intestinal microflora.
Description
Technical field
The present invention relates to a kind of microbe genome DNA indirect extraction method for estimating the animal intestinal Microbial Community Diversity.
Background technology
Exist a large amount of microorganism species in the animal intestinal, these microorganisms have tremendous influence to nutrition, the immunity of body, the aspect such as grow.Their separation and evaluation are for research intestinal microflora function and extremely important with the mutual relationship of body.Traditional method mainly is after enteric microorganism is passed through the selective medium separation and Culture, to carry out the classification of pure bacterium again and identify.This technology is time-consuming, effort not only, but can only detect culturing micro-organisms, and to a large amount of unknown can not culturing micro-organisms helpless in the enteron aisle.Along with the in recent years fast development of modern biotechnology, a large amount of Protocols in Molecular Biologies are applied in the microecology of intestinal tract research, extracting genomic dna from animal intestinal is very useful method, can be used for detecting the microorganism that to cultivate, reach the true mutual relationship between microorganism and host between the reflection enteric microorganism, make us more complete evaluation be arranged to enteric microorganism, also can be used for disclosing the diversity of the gene in the enteric microorganism ecosystem and with the variation of intestinal environment.This just requires extracting and purifying enteric microorganism genomic dna in the driven thing faecal samples.By thereby the research of microbe genome DNA in the sample being understood indirectly wherein distribution and the composition situation of microorganism.And set up efficiently, the microbe genome DNA indirect extraction method also becomes a key problem in technology place of enteric microorganism molecular ecology research accurately.
The major objective of extracting genome DNA is to obtain the highest DNA to reclaim, thereby obtains the most representative DNA from microflora, and carries out further molecule manipulation (such as PCR, SSCP, DGGE, TGGE, AFLP etc.).But the sample from animal intestinal contains very complicated composition, and especially the organic substance such as lipid, acids is difficult to remove in the extracting genome DNA process, directly affects follow-up pcr amplification, hybridization, restriction endonuclease digestion and bacterium conversion etc.In addition, slightly carry each purification step of DNA after the lysis, the repeated purification step that for example carried out before dna molecular research has inevitably caused the loss of DNA.All there is defective in most gene group DNA extraction method, and for example lysis is incomplete, and DNA is adsorbed in the sample top layer, contains losing, degrade and shearing of enzyme inhibitors and DNA in the sample extraction thing.In addition, also mostly continue to use traditional DNA preparation method, i.e. phenol/chloroform extraction method at present.This technological operation step is complicated, and the sample requirement is large, and the DNA yield is low, is difficult to a large amount of samples of fast processing.In addition, the organic solvents such as phenol, chloroform easily cause environmental pollution, diminish operator's health.
Therefore, need research be applicable to convenient, fast, the practical method that the animal intestinal microbial DNA extracts, use DNA sample that ordinary method was obtained to have the PCR inhibition such as acids and the problem such as the lysis rate is low, the DNA loss is serious to solve.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of microbe genome DNA indirect extraction method for estimating the animal intestinal Microbial Community Diversity, utilizes mean density (the 1.1 μ g/cm of bacterium
3) less than mean density (the 1.7-2.3 μ g/cm of faecal samples
3), before lysis, sample is carried out pre-treatment, microorganism cells is separated from their enteron aisle samples, solving after enteric microorganism lysis, there are the problems such as pollutant removal difficulty and the DNA rate of recovery are low in purification step.
In order to achieve the above object, the present invention realizes by the following technical solutions.
Described microbe genome DNA indirect extraction method for estimating the animal intestinal Microbial Community Diversity may further comprise the steps:
1) add the sterilized water mixing of 0.1-10mL precooling in the 0.1-10g animal excrement sample after, then whirlpool concussion 5-20 minute adds 0.1-10mL homogenate buffer A, whirlpool concussion 5-20 minute, room temperature 40-400g is centrifugal, collects supernatant liquor and is transferred in another centrifugal bottle;
2) the homogenate buffer B washing that adds the 0.2-20mL precooling in the throw out that obtains to above-mentioned step 1) is resuspended, whirlpool concussion 5-20 minute, and centrifugal 10 minutes of 50-400g room temperature is got the merging of supernatant liquor and step 1) gained supernatant liquor;
3) with above-mentioned steps 2) under the supernatant liquor room temperature that obtains the centrifugal 10-60 of 6000-15000rpm minute to reclaim somatic cells;
4) reclaim the washings C that adds 1-100ml in the somatic cells that obtains to above-mentioned step 3), washed 2-10 minute, wherein washings C was the trisodium phosphate of 1g/L with the precipitation somatic cells in the centrifugal 10-60 of 6000-15000rpm minute under the room temperature;
5) add the N,O-Diacetylmuramidase of 20-200ul 50mg/ml and the Proteinase K of 10-50ul 20mg/ml in the somatic cells after the precipitation that obtains to above-mentioned step 4), 20-40 ℃ water-bath 10-40 minute, then add 50-500 μ l cell pyrolysis liquid D, put upside down gently mixing 1-10 minute, the centrifugal 5-10 of 6000-15000rpm minute, shard;
6) with above-mentioned steps 5) supernatant that obtains transfers in the clean centrifuge tube, adds 100-1500 μ l albumen and removes liquid E in pipe, gently puts upside down mixing with have gentle hands, and room temperature was placed 5-10 minute, and the centrifugal 3-10 of 8000-15000rpm minute with precipitating proteins;
7) with above-mentioned steps 6) supernatant that obtains transfers in the clean centrifuge tube, adds 2-5 times of volume DNA and connects liquid F in centrifuge tube, puts upside down 1-5 minute mixing with hand;
8) with above-mentioned steps 7) obtain mixed solution and add in the collection tube that contains column, room temperature was placed 1-5 minute, the centrifugal 1-3 of 3000-10000rpm minute, outwell the waste liquid in the collection tube, be reentered in the centrifuge tube in connection with post, it is centrifugal again to add supernatant liquor, until all supernatant liquors are centrifugal complete;
9) with above-mentioned steps 8) collection tube that contains column is placed in the new centrifuge tube, adds 300-700 μ l washings G in column, and 10000-13000rpm is centrifugal, outwells the waste liquid in the collection tube;
10) with above-mentioned steps 9) column relay and reclaim in the collector, and add 400-750 μ l washings H in column, 8000-18000rpm is centrifugal, outwells waste liquid in the collection tube, repeats above-mentioned steps once;
11) with above-mentioned steps 10) column relay the recovery collector, the centrifugal 1-5 of 8000-18000rpm minute;
12) with above-mentioned steps 11) column pack in the new centrifuge tube, room temperature is placed, so that the ethanol volatilization is clean, at the careful 30-50 μ l elutriant I that adds of film central authorities, do not poke film, room temperature is placed, the centrifugal 1-3 of 8000-15000rpm minute, be the DNA of separation in the centrifuge tube, be stored in-20 ℃; Wherein elutriant I is the Tris-HCl of 0.1mM, pH=9.0.
Wherein, in the above-mentioned indirect extraction method, described homogenate buffer A is by 20-400mM Tris, 15-200mM EDTA, 50-350mM NaCl, 0.5-3%pvpp, and 0.1-1% TEEPOL (R) 610S forms, pH=6.0-9.0, and wherein the unit of pvpp is g/L; Preferably form pH=7.5 by 200mM Tris, 100mMEDTA, 200mM NaCl, 2%pvpp, 0.5%TEEPOL (R) 610S.
Described homogenate buffer B is comprised of 10-200mM Tris, 5-100mM EDTA, 25-350mM NaCl, 0.2-1.5%pvpp, and 0.1-1%TEEPOL (R) 610 S form, pH=6.0-9.0; Preferably form pH=7.5 by 100mM Tris, 100mM EDTA, 150mMNaCl, 1%pvpp, 0.5%TEEPOL (R) 610S.
Described cell pyrolysis liquid D is by the 1wt%SDS(sodium lauryl sulphate), 20-60mM Tris, 100-200mM NaCl, 40-100mM EDTA form pH=8.0; Preferably form pH=8.0 by 1wt%SDS, 40mMTris, 150mM NaCl, 60mM EDTA.
Described albumen is removed liquid E and is comprised of 2-5M KAc, 2-8wt% Glacial acetic acid; Preferably formed by 3M KAc, 4wt% Glacial acetic acid.
Described DNA connects liquid F and forms pH=4.5-6.0 by 5-10M guanidinium isothiocyanate, 200-600mM Potassium ethanoate; Preferably form pH=5.5 by 8M guanidinium isothiocyanate, 500mM Potassium ethanoate.
Described washings G is comprised of 6M guanidinium isothiocyanate, 23mM Trisodium Citrate.
Described washings H forms pH=5.0 by 70wt% ethanol, 150mM Potassium ethanoate.
Indirect extraction method of the present invention has the following advantages:
⑴ simple to operate, do not need comparatively complicated equipment, and conventional biological chemistry or microbiology laboratory just can be finished; And the reagent that relates to is the reagent of routine analysis, molecular biology use, and is low with respect to the test kit cost on the market.
⑵ the inventive method is before lysis sample to be carried out pre-treatment, and microorganism cells is separated from their faecal samples, has avoided purification step to have pollutant removal difficulty and the low problem of the DNA rate of recovery.
⑶ the inventive method suitability is strong, the OD of the intestinal microbial DNA of extraction
260/ OD
230And OD
260/ OD
280The value of being near the mark, can directly applying to molecule manipulation, to come evaluate plant roots be Microbial Community Diversity.
⑷ leaching process does not use phenol, chloroform, and the healthy injury, the DNA that obtains that reduce the experimenter are complete, and molecule fragment is greater than 10kb, and output is high.
Embodiment
Below in conjunction with specific embodiment technical scheme of the present invention is described in further detail.
The EDTA(ethylenediamine tetraacetic acid (EDTA)), Tris(tricarboxylic ylmethyl aminomethane), SDS(sodium lauryl sulphate), NaCl, ethanol, trisodium phosphate, pvpp(cross-linked polyvinylpyrrolidone), Tai-Ace S 150, guanidinium isothiocyanate, KAc(Potassium ethanoate), the reagent such as Glacial acetic acid, Potassium ethanoate, Trisodium Citrate is domestic analytical pure medicine.TEEPOL (R) 610S(washing composition) available from SPECTRUM-USP company.
N,O-Diacetylmuramidase and Proteinase K are that worker's import packing is given birth in Shanghai.
Embodiment 1
1) behind the sterilized water mixing of 0.5g test with adding 0.5mL precooling in the small white mouse animal excrement sample, then whirlpool concussion 10 minutes adds 0.5mL homogenate buffer A, whirlpool concussion 10 minutes, centrifugal 10 minutes of low speed (250g) room temperature is collected supernatant liquor and is transferred in another centrifugal bottle.
2) it is resuspended upwards to go on foot the homogenate buffer B washing that adds the 1mL precooling in the throw out of gained, whirlpool concussion 10 minutes, centrifugal 10 minutes of low speed (250g) room temperature, get supernatant liquor, merge with step 1) gained supernatant liquor, 10000rpm abandoned supernatant to reclaim somatic cells in centrifugal 30 minutes under the room temperature.
3) upwards go on foot the washings C that adds 1.5ml in the somatic cells of gained, wash 5 minutes, centrifugal 30 minutes of 10000rpm is to precipitate somatic cells under the room temperature.Add 160ul N,O-Diacetylmuramidase (50mg/ml) and 20ul Proteinase K (20mg/ml) in the somatic cells after the precipitation, 37 ℃ water-bath 30-40 minute, then add 122 μ l cell pyrolysis liquid D, whirlpool shook 5-20 minute.Centrifugal 10 minutes of 13000rpm, shard.
4) supernatant of gained of upper step is transferred in the clean centrifuge tube, added 250 μ l albumen and remove liquid E in pipe, gently put upside down mixing 10 times with have gentle hands, room temperature was placed 5 minutes, centrifugal 5 minutes of 13000rpm.
5) supernatant of gained of upper step is transferred in the clean centrifuge tube, added 3 times of volume DNA and connect liquid F in centrifuge tube, put upside down mixing 1-5 minute with hand.
6) one of mixed solution adding with gained of upper step contains in the collection tube of column, and room temperature was placed 2 minutes.Centrifugal 1 minute of 6000rpm outwells the waste liquid in the collection tube, is reentered in the centrifuge tube in connection with post, and it is centrifugal again to add supernatant liquor, until all supernatant liquors are centrifugal complete.
7) the above-mentioned collection tube that contains column is placed in the new centrifuge tube, adds 500 μ l washings G in column, centrifugal 1 minute of 13000rpm outwells the waste liquid in the collection tube.
8) relay in the recovery collector in connection with post, and add 650 μ l washings H in column, centrifugal 1 minute of 13000rpm outwells the waste liquid in the collection tube, repeats above-mentioned steps once.
9) relay the recovery collector in connection with post, centrifugal 2 minutes of 13000rpm.
10) pack in the new centrifuge tube in connection with post, room temperature was placed several minutes, so that the ethanol volatilization is clean, at the careful 30 μ l elutriant I that add of film central authorities.Do not poke film, room temperature was placed 2 minutes.Centrifugal 1 minute of 13000rpm is the DNA of separation in the centrifuge tube, be stored in-20 ℃.
The add-on of above solution all can be according to the increase of the amount of extracting sample, and proportional increase.
Use spectrophotometric determination OD
230, OD
260And OD
280, the result shows OD
260/ OD
230=2.01, OD
260/ OD
280=1.80, (annotate: OD close to standard value
260/ OD
230Standard value is 2.0, OD
260/ OD
280Standard value is 1.8), can directly apply to molecule manipulation, estimate the animal intestinal Microbial Community Diversity.
Embodiment 2
1) behind the sterilized water mixing of 0.5g test with adding 0.5mL precooling in the rat faecal samples, then whirlpool concussion 10 minutes adds 0.5mL homogenate buffer A, whirlpool concussion 10 minutes, centrifugal 10 minutes of low speed (250g) room temperature is collected supernatant liquor and is transferred in another centrifugal bottle.
2) it is resuspended upwards to go on foot the homogenate buffer B washing that adds the 1mL precooling in the throw out of gained, whirlpool concussion 10 minutes, centrifugal 10 minutes of low speed (250g) room temperature, get supernatant liquor, merge with step 1) gained supernatant liquor, 10000rpm abandoned supernatant to reclaim somatic cells in centrifugal 30 minutes under the room temperature.
3) upwards go on foot the washings C that adds 1.5ml in the somatic cells of gained, wash 5 minutes, centrifugal 30 minutes of 10000rpm is to precipitate somatic cells under the room temperature.Add 160ul N,O-Diacetylmuramidase (50mg/ml) and 20ul Proteinase K (20mg/ml) in the somatic cells after the precipitation, 37 ℃ water-bath 30-40 minute, then add 122 μ l cell pyrolysis liquid D, whirlpool shook 5-20 minute.Centrifugal 10 minutes of 13000rpm, shard.
4) supernatant of gained of upper step is transferred in the clean centrifuge tube, added 250 μ l albumen and remove liquid E in pipe, gently put upside down mixing 10 times with have gentle hands, room temperature was placed 5 minutes, centrifugal 5 minutes of 13000rpm.
5) supernatant of gained of upper step is transferred in the clean centrifuge tube, added 3 times of volume DNA and connect liquid F in centrifuge tube, put upside down mixing 1-5 minute with hand.
6) one of mixed solution adding with gained of upper step contains in the collection tube of column, and room temperature was placed 2 minutes.Centrifugal 1 minute of 6000rpm outwells the waste liquid in the collection tube, is reentered in the centrifuge tube in connection with post, and it is centrifugal again to add supernatant liquor, until all supernatant liquors are centrifugal complete.
7) the above-mentioned collection tube that contains column is placed in the new centrifuge tube, adds 500 μ l washings G in column, centrifugal 1 minute of 13000rpm outwells the waste liquid in the collection tube.
8) above-mentioned column is relay in the recovery collector, and add 650 μ l washings H in column, centrifugal 1 minute of 13000rpm outwells waste liquid in the collection tube, repeats above-mentioned steps once.
9) relay the recovery collector in connection with post, centrifugal 2 minutes of 13000rpm.
10) pack in the new centrifuge tube in connection with post, room temperature was placed several minutes, so that the ethanol volatilization is clean, at the careful 30 μ l elutriant I that add of film central authorities.Do not poke film, room temperature was placed 2 minutes.Centrifugal 1 minute of 13000rpm is the DNA of separation in the centrifuge tube, be stored in-20 ℃.
The add-on of above solution all can be according to the increase of the amount of extracting sample, and proportional increase.
Use spectrophotometric determination OD
230, OD
260And OD
280, the result shows OD
260/ OD
230=2.03, OD
260/ OD
280=1.82, (annotate: OD close to standard value
260/ OD
230Standard value is 2.0, OD
260/ OD
280Standard value is 1.8), can directly apply to molecule manipulation, estimate the animal intestinal Microbial Community Diversity.
Embodiment 3
1) behind the sterilized water mixing of 0.5g test with adding 0.5mL precooling in the white rabbit faecal samples, then whirlpool concussion 10 minutes adds 0.5mL homogenate buffer A, whirlpool concussion 10 minutes, centrifugal 10 minutes of low speed (250g) room temperature is collected supernatant liquor and is transferred in another centrifugal bottle.
2) it is resuspended upwards to go on foot the homogenate buffer B washing that adds the 1mL precooling in the throw out of gained, whirlpool concussion 10 minutes, centrifugal 10 minutes of low speed (250g) room temperature, get supernatant liquor, merge with step 1) gained supernatant liquor, 10000rpm abandoned supernatant to reclaim somatic cells in centrifugal 30 minutes under the room temperature.
3) upwards go on foot the washings C that adds 1.5ml in the somatic cells of gained, wash 5 minutes, centrifugal 30 minutes of 10000rpm is to precipitate somatic cells under the room temperature.Add 160ul N,O-Diacetylmuramidase (50mg/ml) and 20ul Proteinase K (20mg/ml) in the somatic cells after the precipitation, 37 ℃ water-bath 30-40 minute, then add 122 μ l cell pyrolysis liquid D, whirlpool shook 5-20 minute.Centrifugal 10 minutes of 13000rpm, shard.
4) supernatant of gained of upper step is transferred in the clean centrifuge tube, added 250 μ l albumen and remove liquid E in pipe, gently put upside down mixing 10 times with have gentle hands, room temperature was placed 5 minutes, centrifugal 5 minutes of 13000rpm.
5) supernatant of gained of upper step is transferred in the clean centrifuge tube, added 3 times of volume DNA and connect liquid F in centrifuge tube, put upside down mixing 1-5 minute with hand.
6) in the mixed solution of gained of upper step being added in the collection tube that contains column, room temperature was placed 2 minutes.Centrifugal 1 minute of 6000rpm outwells the waste liquid in the collection tube, is reentered in the centrifuge tube in connection with post, and it is centrifugal again to add supernatant liquor, until all supernatant liquors are centrifugal complete.
7) the above-mentioned collection tube that contains column is placed in the new centrifuge tube, adds 500 μ l washings G in column, centrifugal 1 minute of 13000rpm outwells the waste liquid in the collection tube.
8) above-mentioned column is relay in the recovery collector, and add 650 μ l washings H in column, centrifugal 1 minute of 13000rpm outwells waste liquid in the collection tube, repeats above-mentioned steps once.
9) relay the recovery collector in connection with post, centrifugal 2 minutes of 13000rpm.
10) pack in the new centrifuge tube in connection with post, room temperature was placed several minutes, so that the ethanol volatilization is clean, at the careful 30 μ l elutriant I that add of film central authorities.Do not poke film, room temperature was placed 2 minutes.Centrifugal 1 minute of 13000rpm is the DNA of separation in the centrifuge tube, be stored in-20 ℃.
The add-on of above solution all can be according to the increase of the amount of extracting sample, and proportional increase.
Use spectrophotometric determination OD
230, OD
260And OD
280, the result shows OD
260/ OD
230=2.03, OD
260/ OD
280=1.78, (annotate: OD close to standard value
260/ OD
230Standard value is 2.0, OD
260/ OD
280Standard value is 1.8), can directly apply to molecule manipulation, estimate the animal intestinal Microbial Community Diversity.
Claims (8)
1. a microbe genome DNA indirect extraction method that is used for estimating the animal intestinal Microbial Community Diversity is characterized in that, may further comprise the steps:
1) add the sterilized water mixing of 0.1-10mL precooling in the 0.1-10g animal excrement sample after, then whirlpool concussion 5-20 minute adds 0.1-10mL homogenate buffer A, whirlpool concussion 5-20 minute, room temperature 40-400g is centrifugal, collects supernatant liquor and is transferred in another centrifugal bottle;
2) the homogenate buffer B washing that adds the 0.2-20mL precooling in the throw out that obtains to above-mentioned step 1) is resuspended, whirlpool concussion 5-20 minute, and room temperature 50-400g is centrifugal, gets the merging of supernatant liquor and step 1) gained supernatant liquor;
3) with above-mentioned steps 2) under the supernatant liquor room temperature that obtains the centrifugal 10-60 of 6000-15000rpm minute to reclaim somatic cells;
4) reclaim the washings C that adds 1-100ml in the somatic cells that obtains to above-mentioned step 3), washed 2-10 minute, wherein washings C was the trisodium phosphate of 1g/L with the precipitation somatic cells in the centrifugal 10-60 of 6000-15000rpm minute under the room temperature;
5) add the N,O-Diacetylmuramidase of 20-200ul 50mg/ml and the Proteinase K of 10-50ul 20mg/ml in the somatic cells after the precipitation that obtains to above-mentioned step 4), 20-40 ℃ water-bath 10-40 minute, then add 50-500 μ l cell pyrolysis liquid D, put upside down gently mixing 1-10 minute, the centrifugal 5-10 of 6000-15000rpm minute, shard;
6) with above-mentioned steps 5) the centrifugal supernatant that obtains transfers in the clean centrifuge tube, add 100-1500 μ l albumen and remove liquid E in pipe, gently put upside down mixing with have gentle hands, room temperature was placed 5-10 minute, and the centrifugal 3-10 of 8000-15000rpm minute with precipitating proteins;
7) with above-mentioned steps 6) supernatant that obtains transfers in the clean centrifuge tube, adds 2-5 times of volume DNA and connects liquid F in centrifuge tube, puts upside down 1-5 minute mixing with hand;
8) with above-mentioned steps 7) obtain mixed solution and add in the collection tube that contains column, room temperature was placed 1-5 minute, the centrifugal 1-3 of 3000-10000rpm minute, outwell the waste liquid in the collection tube, be reentered in the centrifuge tube in connection with post, it is centrifugal again to add supernatant liquor, until all supernatant liquors are centrifugal complete;
9) with above-mentioned steps 8) contain column collection tube be placed in the new centrifuge tube, add 300-700 μ l washings G in column, 10000-13000rpm is centrifugal, outwells the waste liquid in the collection tube;
10) with above-mentioned steps 9) column relay and reclaim in the collector, and add 400-750 μ l washings H in column, 8000-18000rpm is centrifugal, outwells waste liquid in the collection tube, repeats above-mentioned steps once;
11) with above-mentioned steps 10) column relay the recovery collector, the centrifugal 1-5 of 8000-18000rpm minute;
12) with above-mentioned steps 11) column pack in the new centrifuge tube, room temperature is placed, so that the ethanol volatilization is clean, at the careful 30-50 μ l elutriant I that adds of film central authorities, do not poke film, room temperature is placed, the centrifugal 1-3 of 8000-15000rpm minute, be the DNA of separation in the centrifuge tube, be stored in-20 ℃; Wherein elutriant I is the Tris-HCl of 0.1mM, pH=9.0.
2. extracting method according to claim 1, it is characterized in that described homogenate buffer A is by 20-400mM Tris, 15-200mM EDTA, 50-350mM NaCl, 0.5-3%pvpp, 0.1-1%TEEPOL (R) 610S forms, pH=6.0-9.0, wherein the unit of pvpp is g/L.
3. extracting method according to claim 1, it is characterized in that, described homogenate buffer B is that 10-200mM Tris, 5-100mM EDTA, 25-350mM NaCl, 0.2-1.5%pvpp form, and 0.1-1%TEEPOL (R) 610S forms, pH=6.0-9.0.
4. extracting method according to claim 1 is characterized in that, described cell pyrolysis liquid D forms pH=8.0 by 1wt%SDS, 20-60mM Tris, 100-200mM NaCl, 40-100mM EDTA.
5. extracting method according to claim 1 is characterized in that, described albumen is removed liquid E and is comprised of 2-5M KAc, 2-8wt% Glacial acetic acid.
6. extracting method according to claim 1 is characterized in that, described DNA connects liquid F and forms pH=4.5-6.0 by 5-10M guanidinium isothiocyanate, 200-600mM Potassium ethanoate.
7. extracting method according to claim 1 is characterized in that, described washings G is comprised of 6M guanidinium isothiocyanate, 23mM Trisodium Citrate.
8. extracting method according to claim 1 is characterized in that, described washings H forms pH=5.0 by 70% ethanol, 150mM Potassium ethanoate.
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| CN101307311A (en) * | 2008-07-14 | 2008-11-19 | 四川大学 | Process for abstracting total DNA of swine waste sample |
| CN101831499A (en) * | 2010-05-24 | 2010-09-15 | 东北农业大学 | Real-time PCR method for detecting establishment number of oxalobacter formigens in excrement |
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| CN108034652A (en) * | 2017-12-18 | 2018-05-15 | 浙江省农业科学院 | The extracting method of microorganism total DNA in a kind of animal and bird intestines |
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