CN101392248A - Extraction method of anaerobic activated sludge DNA - Google Patents
Extraction method of anaerobic activated sludge DNA Download PDFInfo
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- CN101392248A CN101392248A CNA2008100647711A CN200810064771A CN101392248A CN 101392248 A CN101392248 A CN 101392248A CN A2008100647711 A CNA2008100647711 A CN A2008100647711A CN 200810064771 A CN200810064771 A CN 200810064771A CN 101392248 A CN101392248 A CN 101392248A
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Abstract
The invention discloses a method for extracting the DNA of anaerobic activated sludge and relates to a method for extracting the DNA of activated sludge. The invention solves the defects of small yield and low purity rate existing in the existing method for extracting the DNA of the anaerobic activated sludge. The method for extracting the DNA of the anaerobic activated sludge, provided by the invention, is carried out according to the steps as follows: firstly, sludge cleaning; secondly, cell decomposition; thirdly, purification of the DNA; and then the DNA of the anaerobic activated sludge is extracted. The DNA extracted by the DNA extraction method of the invention has high purity rate and large yield.
Description
Technical field
The present invention relates to the extracting method of activated sludge DNA.
Background technology
At present, adopt enzyme process (as N,O-Diacetylmuramidase, Proteinase K etc.) or test kit method to extract DNA usually.But, in the anaerobic activated sludge DNA leaching process, find, suspended substance in the mud, colloidalmaterial, sulfide, soil ulmin etc. have inhibiting composition to the activity of N,O-Diacetylmuramidase, Proteinase K and Taq enzyme, the DNA amount that causes extracting less, purity is not enough, can not reflect diversity and the population abundance of microorganism in the anaerobic activated sludge exactly.If the high effect that can influence the PCR reaction of inhibitor contents causes the failure that yields poorly even increase of PCR product, become the rate-limiting step of restriction follow-up denaturing gradient gel electrophoresis (DGGE) technology, the operation of single strand conformation polymorphism (SSCP) technology equimolecular.
Summary of the invention
There is the defective that output is little, purity is low in the present invention in order to solve the existing method of extracting anaerobic activated sludge DNA, and a kind of extracting method of anaerobic activated sludge DNA is provided.
The present invention extracts the method for anaerobic activated sludge DNA and carries out as follows:
One, mud cleans:
A, the anaerobic activated sludge of getting 2g add in the centrifuge tube of 1.5mL, add the PBS damping fluid concussion of 1mL, with the centrifugal 1min of the speed of 9000r/min;
B, get centrifugal lower floor mud, repeating step a twice promptly obtains cleaned anaerobic activated sludge;
Two, lysing cell:
Get the cleaned anaerobic activated sludge of 100mg and put into the centrifuge tube of 1.5mL, add 0.5g quartz sand, 40 μ L DNA extraction damping fluids, 20 μ L concentration are that RNA enzyme and the 160 μ L concentration of 40mg/mL are the N,O-Diacetylmuramidase of 100mg/mL, in 37 ℃ of insulation 1h, and put upside down centrifuge tube back and forth 20～30 times, add 400 μ L DNA extraction damping fluids again, 40 μ L concentration are Proteinase K and the 600 μ L SDS damping fluids of 100mg/mL, in 60 ℃ of insulation 1h, and put upside down centrifuge tube back and forth 20～30 times, with the centrifugal 1min of the speed of 9000r/min, get supernatant liquor and add in another centrifuge tube;
Three, the purifying of DNA:
The Virahol that in the supernatant liquor that step 2 obtains, adds supernatant liquor volume 1/4, join in the centrifugal adsorption column, with the centrifugal 15s of the speed of 9000r/min, outwell the liquid in the collection tube, again with the centrifugal 15s of the speed of 9000r/min, adding 500 μ L mass concentrations are 75% ethanol in centrifugal adsorption column, leave standstill 1min, to outwell the liquid in the collection tube behind the centrifugal 30s of the speed of 9000r/min, again centrifugal adsorption column is put into same collection tube, add 500 μ L mass concentrations again and be 75% ethanol, with the centrifugal 30s of 9000r/min, outwell the liquid in the collection tube, centrifugal adsorption column is put into collection tube again with the centrifugal 1min of the speed of 9000r/min, then centrifugal adsorption column is transferred in the new collection tube, the Tris solution (tris solution) that with 100 μ L concentration is 2mmol/L then joins in the centrifugal adsorption column, under 60 ℃ condition, be incubated 5min,, promptly extract the DNA of anaerobic activated sludge again with the centrifugal 1min of the speed of 12000r/min.
The pH value of the PBS damping fluid in the extracting method step 1 of anaerobic activated sludge DNA of the present invention is 7.4, and the mass concentration of NaCl is 0.8% in the PBS damping fluid, the mass concentration of KCl is 0.02%, Na
2HPO
4Mass concentration be 0.14%, KH
2PO
4Mass concentration be 0.02%; The pH value of the DNA extraction damping fluid in the step 2 is 8.0, and the concentration of Tris-HCl is 100mmol/L in the DNA extraction damping fluid, and the concentration of EDTA is 100mmol/L, and the concentration of NaCl is 200mmol/L, and the mass concentration of CTAB is 2.0%; The pH value of the SDS damping fluid in the step 2 is 8.0, and the concentration of Tris-HCl is 100mmol/L in the SDS damping fluid, and the concentration of NaCl is 200mmol/L, and the mass concentration of SDS is 2.0%.
The principle of the extracting method of anaerobic activated sludge DNA of the present invention and advantage: 1, reduced the content of detesting inhibition in the supporting property mud, helped follow-up lysis, effectively increased output and the purity of extracting dna profiling by the mud cleaning; 2, lysis adopts the method for enzyme process and the coupling of physics method to increase the output of dna profiling; 3, the phenomenon that DNA runs off in the adsorption column purifying purge process rationally having avoided too much causing owing to impurity has effectively increased dna profiling output and the purity extracted; 4, in sum, the existing method of the rate ratio of the dna profiling that the present invention extracts has improved 200%～400%, and the purity of dna profiling has improved 38.8%～46.2% than existing method.
Description of drawings
Fig. 1 is that the anaerobic activated sludge DNA of embodiment five extractions and the anaerobic activated sludge DNA agarose of existing method extraction detect comparison diagram, and Fig. 2 is that the anaerobic activated sludge DNA of embodiment five extractions and anaerobic activated sludge DNA denaturing gradient gel electrophoresis (DGGE) the test sample microbial diversity of existing method extraction contrast collection of illustrative plates.
Embodiment
Embodiment one: present embodiment is extracted the method for anaerobic activated sludge DNA and is carried out as follows:
One, mud cleans:
A, the anaerobic activated sludge of getting 2g add in the centrifuge tube of 1.5mL, add the PBS damping fluid concussion of 1mL, with the centrifugal 1min of the speed of 9000r/min;
B, get centrifugal lower floor mud, repeating step a twice promptly obtains cleaned anaerobic activated sludge;
Two, lysing cell:
Get the cleaned anaerobic activated sludge of 100mg and put into the centrifuge tube of 1.5mL, add 0.5g quartz sand, 40 μ L DNA extraction damping fluids, 20 μ L concentration are that RNA enzyme and the 160 μ L concentration of 40mg/mL are the N,O-Diacetylmuramidase of 100mg/mL, in 37 ℃ of insulation 1h, and put upside down centrifuge tube back and forth 20～30 times, add 400 μ LDNA again and extract damping fluid, 40 μ L concentration are Proteinase K and the 600 μ L SDS damping fluids of 100mg/mL, in 60 ℃ of insulation 1h, and put upside down centrifuge tube back and forth 20～30 times, with the centrifugal 1min of the speed of 9000r/min, get supernatant liquor and add in another centrifuge tube;
Three, the purifying of DNA:
The Virahol that in the supernatant liquor that step 2 obtains, adds supernatant liquor volume 1/4, join in the centrifugal adsorption column, with the centrifugal 15s of the speed of 9000r/min, outwell the liquid in the collection tube, again with the centrifugal 15s of the speed of 9000r/min, adding 500 μ L mass concentrations are 75% ethanol in centrifugal adsorption column, leave standstill 1min, to outwell the liquid in the collection tube behind the centrifugal 30s of the speed of 9000r/min, again centrifugal adsorption column is put into same collection tube, add 500 μ L mass concentrations again and be 75% ethanol, with the centrifugal 30s of 9000r/min, outwell the liquid in the collection tube, centrifugal adsorption column is put into collection tube again with the centrifugal 1min of the speed of 9000r/min, then centrifugal adsorption column is transferred in the new collection tube, the Tris solution (tris solution) that with 100 μ L concentration is 2mmol/L then joins in the centrifugal adsorption column, under 60 ℃ condition, be incubated 5min,, promptly extract the DNA of anaerobic activated sludge again with the centrifugal 1min of the speed of 12000r/min.
Adsorption column, RNA enzyme, N,O-Diacetylmuramidase, Proteinase K and Proteinase K lysate used in the present embodiment are all available from Shanghai China Shun biotechnology company limited.
Embodiment two: the difference of present embodiment and embodiment one is: the pH value of the PBS damping fluid in the step 1 is 7.4, and the mass concentration of NaCl is 0.8% in the PBS damping fluid, the mass concentration of KCl is 0.02%, Na
2HPO
4Mass concentration be 0.14%, KH
2PO
4Mass concentration be 0.02%.Other step and parameter are identical with embodiment one.
Embodiment three: the difference of present embodiment and embodiment one is: the pH value of the DNA extraction damping fluid in the step 2 is 8.0, the concentration of Tris-HCl is 100mmol/L in the DNA extraction damping fluid, the concentration of EDTA is 100mmol/L, the concentration of NaCl is 200mmol/L, and the mass concentration of CTAB is 2.0%.Other step and parameter are identical with embodiment one.
Embodiment four: the difference of present embodiment and embodiment one is: the pH value of the SDS damping fluid in the step 2 is 8.0, the concentration of Tris-HCl is 100mmol/L in the SDS damping fluid, the concentration of NaCl is 200mmol/L, and the mass concentration of SDS is 2.0%.Other step and parameter are identical with embodiment one.
Embodiment five: the difference of present embodiment and embodiment one is: the pH value of the PBS damping fluid in the step 1 is 7.4, and the mass concentration of NaCl is 0.8% in the PBS damping fluid, the mass concentration of KCl is 0.02%, Na
2HPO
4Mass concentration be 0.14%, KH
2PO
4Mass concentration be 0.02%; The pH value of the DNA extraction damping fluid in the step 2 is 8.0, and the concentration of Tris-HCl is 100mmol/L in the DNA extraction damping fluid, and the concentration of EDTA is 100mmol/L, and the concentration of NaCl is 200mmol/L, and the mass concentration of CTAB is 2.0%; The pH value of the SDS damping fluid in the step 2 is 8.0, and the concentration of Tris-HCl is 100mmol/L in the SDS damping fluid, and the concentration of NaCl is 200mmol/L, and the mass concentration of SDS is 2.0%.Other step and parameter are identical with embodiment one.
The DNA that DNA that the method steps three of present embodiment is extracted and following existing method are extracted contrasts:
Adopt the method for present embodiment to make two parallel samples, be numbered 3-a, 3-b; Adopt Enzymatic Extraction DNA to make two parallel samples, be numbered 1-a, 1-b; Adopt the test kit method to extract DNA and make two parallel samples, be numbered 2-a, 2-b; The DGGE that above-mentioned parallel sample is carried out agarose detection and DNA sample microbial diversity respectively detects, and the agarose detected result as shown in Figure 1; DGGE detects DNA sample microbial diversity collection of illustrative plates as shown in Figure 2; The A260/A280 value and the DGGE collection of illustrative plates band number that characterize DNA purity are as shown in table 1.
The purity of table 1 DNA extraction and DGGE band number
| 1-a | 1-b | 2-a | 2-b | 3-a | 3-b | |
| A260/A280 | 1.288 | 1.276 | 1.408 | 1.401 | 1.674 | 1.580 |
| DGGE band number | 8 | 9 | 13 | 13 | 17 | 17 |
The purity of the dna profiling that table 1 explanation present embodiment is extracted has improved 38.8%～46.2% than existing method.
The existing method of the rate ratio of the dna profiling of present embodiment extraction obviously improves as can be seen from Figure 1.
From Fig. 2 associative list 1 as can be seen the dna profiling that extracts of present embodiment more can embody the diversity of microorganism than existing method because its DGGE band number is Duoed 4 to 8 than existing method.
Claims (4)
1, a kind of extracting method of anaerobic activated sludge DNA is characterized in that the method for extracting anaerobic activated sludge DNA carries out as follows:
One, mud cleans:
A, the anaerobic activated sludge of getting 2g add in the centrifuge tube of 1.5mL, add the PBS damping fluid concussion of 1mL, with the centrifugal 1min of the speed of 9000r/min;
B, get centrifugal lower floor mud, repeating step a twice promptly obtains cleaned anaerobic activated sludge;
Two, lysing cell:
Get the cleaned anaerobic activated sludge of 100mg and put into the centrifuge tube of 1.5mL, add 0.5g quartz sand, 40 μ L DNA extraction damping fluids, 20 μ L concentration are that RNA enzyme and the 160 μ L concentration of 40mg/mL are the N,O-Diacetylmuramidase of 100mg/mL, in 37 ℃ of insulation 1h, and put upside down centrifuge tube back and forth 20～30 times, add 400 μ L DNA extraction damping fluids again, 40 μ L concentration are Proteinase K and the 600 μ L SDS damping fluids of 100mg/mL, in 60 ℃ of insulation 1h, and put upside down centrifuge tube back and forth 20～30 times, with the centrifugal 1min of the speed of 9000r/min, get supernatant liquor and add in another centrifuge tube;
Three, the purifying of DNA:
The Virahol that in the supernatant liquor that step 2 obtains, adds supernatant liquor volume 1/4, join in the centrifugal adsorption column, with the centrifugal 15s of the speed of 9000r/min, outwell the liquid in the collection tube, again with the centrifugal 15s of the speed of 9000r/min, adding 500 μ L mass concentrations are 75% ethanol in centrifugal adsorption column, leave standstill 1min, to outwell the liquid in the collection tube behind the centrifugal 30s of the speed of 9000r/min, again centrifugal adsorption column is put into same collection tube, add 500 μ L mass concentrations again and be 75% ethanol, with the centrifugal 30s of 9000r/min, outwell the liquid in the collection tube, centrifugal adsorption column is put into collection tube again with the centrifugal 1min of the speed of 9000r/min, then centrifugal adsorption column is transferred in the new collection tube, the Tris solution that with 100 μ L concentration is 2mmol/L then joins in the centrifugal adsorption column, under 60 ℃ condition, be incubated 5min,, promptly extract the DNA of anaerobic activated sludge again with the centrifugal 1min of the speed of 12000r/min.
2, the extraction and purification method of a kind of anaerobic activated sludge DNA according to claim 1, the pH value that it is characterized in that the PBS damping fluid in the step 1 is 7.4, the mass concentration of NaCl is 0.8% in the PBS damping fluid, the mass concentration of KCl is 0.02%, Na
2HPO
4Mass concentration be 0.14%, KH
2PO
4Mass concentration be 0.02%.
3, the extraction and purification method of a kind of anaerobic activated sludge DNA according to claim 1, the pH value that it is characterized in that the DNA extraction damping fluid in the step 2 is 8.0, the concentration of Tris-HCl is 100mmol/L in the DNA extraction damping fluid, the concentration of EDTA is 100mmol/L, the concentration of NaCl is 200mmol/L, and the mass concentration of CTAB is 2.0%.
4, the extracting method of a kind of anaerobic activated sludge DNA according to claim 1, the pH value that it is characterized in that the SDS damping fluid in the step 2 is 8.0, the concentration of Tris-HCl is 100mmol/L in the SDS damping fluid, and the concentration of NaCl is 200mmol/L, and the mass concentration of SDS is 2.0%.
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| CN102146363A (en) * | 2010-02-10 | 2011-08-10 | 中国科学院上海生命科学研究院 | Novel glucanase, and coding gene and application thereof |
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| CN102146368A (en) * | 2010-02-10 | 2011-08-10 | 中国科学院上海生命科学研究院 | Method for obtaining biomass conversion related genes from anaerobic fermentation system |
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| CN102161987B (en) * | 2010-02-20 | 2013-03-27 | 大连水产学院 | Method for extracting genomic deoxyribonucleic aid (DNA) of sediment microorganisms in mariculture pond |
| CN103789300A (en) * | 2014-02-26 | 2014-05-14 | 济南大学 | Extraction method of metagenome DNA (deoxyribonucleic acid) in activated sludge for treating epoxypropane saponification wastewater |
| CN103789300B (en) * | 2014-02-26 | 2015-08-12 | 济南大学 | A kind of extracting method of epoxy propane saponified wastewater active sludge macro genome DNA |
| CN105985948A (en) * | 2015-02-28 | 2016-10-05 | 天津市农业质量标准与检测技术研究所 | Method for extracting animal muscular tissue DNA efficiently and safely |
| CN104911178A (en) * | 2015-06-19 | 2015-09-16 | 厦门大学 | Method for simultaneously extracting microbial intracellular and extracellular DNAs (deoxyribonucleic acids) in sewage biological treatment water sample |
| CN109810951A (en) * | 2019-04-09 | 2019-05-28 | 浙江大学 | A method of the Extraction and enrichment bacteriophage from activated sludge |
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