CN103710337B - A kind of method of high efficiency extraction anaerobic grain sludge microorganism total DNA - Google Patents
A kind of method of high efficiency extraction anaerobic grain sludge microorganism total DNA Download PDFInfo
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Abstract
The invention belongs to environmental molecules biology techniques field, be specifically related to a kind of method of high efficiency extraction anaerobic grain sludge microorganism total DNA, step is as follows: thalline is centrifugal; Add DNA stock solution I, N,O-Diacetylmuramidase incubation after solids DNA stock solution I washes, add stock solution II, stock solution III, stock solution IV incubation; Add phenol, chloroform and primary isoamyl alcohol mixed solution, soft 8 word mixings; Centrifuging and taking supernatant, adds chloroform and primary isoamyl alcohol mixed solution, soft 8 word mixings; Centrifuging and taking supernatant, adds Virahol, softly mixes ,-20 DEG C, leaves standstill 30min; Room temperature is centrifugal; 75% ethanol washes one time, room temperature centrifugal 12000r/min, 5min; Add stock solution V resuspended, 4 DEG C of dissolvings of spending the night, obtain anaerobic grain sludge microorganism total DNA.This invention simplifies microorganism total DNA extraction step, achieve the microorganism total DNA extracting anaerobic grain sludge fast and efficiently.
Description
Technical field
The invention belongs to environmental molecules biology techniques field, be specifically related to a kind of method of high efficiency extraction anaerobic grain sludge microorganism total DNA.
Background technology
Granule sludge is owing to having good settling property, and the advantages such as volumetric loading is high, and capacity of resisting impact load is strong, have become the main body of hard-degraded substance biological wastewater treatment at present.Microflora in research granule sludge for inquiring into the reaction mechanism of wastewater treatment, process modification, optimal control all have important effect.But, due to the restriction of traditional analytical procedure, biological community structure and spatial distribution fully can not be disclosed for information about.At present, group's composition, structure diversity and the Plant population change of microorganism effectively can be analyzed by Protocols in Molecular Biology.And most of molecular biology experiment is all premised on the genome DNA extracting research object, therefore, set up efficient, reliable microorganism total DNA extracting method and just seem particularly important.
About the existing a large amount of report of the method extracting microbial DNA from water sample, soil, active sludge and oceanic sediment, but the method extracting DNA from anaerobic grain sludge is also rarely found.
Up to now, less to the research of active sludge sample DNA extracting method both at home and abroad, and mostly study only fewer counting method, cogency is not strong.In addition, also objection is existed to the suitability of certain methods, as Aguilera etc. finds for the acidophilic microbes in the middle of settling, use pearl mill homogenate method can obtain the high lysis rate of more than 95%; And Zheng Xuesong etc. cry the microorganism found for active sludge, the DNA output that pearl mill homogenate method obtains is minimum, and containing a large amount of protein and phenol impurity.Due in granule sludge except the micropopulation had with metabolic activity is external, also there is the residue of endogenous metabolism, autoxidation; The materials such as the organism of the difficult degradation that sewage is brought into and inorganics, the humic substances wherein existed etc. easily disturb the detection of DNA, these pollute the activity of the Taq DNA polymerase in possibility suppression PCR, the digestion of interference restriction enzyme, reduce the hybrid specificities etc. of transformation efficiency and DNA, therefore highly purified DNA is to follow-up work important.
But the method for the anaerobic grain sludge extraction microorganism total DNA of report has a lot of shortcoming at present, as: the DNA extraction total amount of extraction is few, purity is low, containing PCR enzyme inhibitors, and complex steps.
Summary of the invention
The object of this invention is to provide a kind of method that microorganism total DNA that can be used for anaerobic grain sludge extracts.The method has that DNA extraction efficiency is high, purity is high, not containing advantages such as PCR enzyme inhibitorss.
For achieving the above object, the present invention adopts following technical proposals:
Extract a method for anaerobic grain sludge microorganism total DNA, comprise the following steps:
1) thalline is centrifugal,
Measure the thalline of certain volume, put into centrifuge; Discard supernatant, bottom solids DNA stock solution I washes 2 times; Add DNA stock solution I, mixing;
2) add N,O-Diacetylmuramidase, mixing, 30 ~ 40 DEG C of incubation 0.5 ~ 2h, add stock solution II, mixing, 30 ~ 40 DEG C of incubation 0.5 ~ 2h;
3) stock solution III is added, mixing;
4) stock solution IV is added, mixing, 60 ~ 70 DEG C of incubation 5 ~ 15min;
5) phenol, chloroform and primary isoamyl alcohol mixed solution is added, soft 8 word mixing 5 ~ 15min; Room temperature leaves standstill 5 ~ 15min; Centrifugal;
6) get supernatant, add chloroform and primary isoamyl alcohol mixed solution, soft 8 word mixing 5 ~ 15min; Room temperature leaves standstill 5 ~ 15min; Centrifugal;
7) get supernatant, add Virahol, softly mix ,-20 DEG C, leave standstill 30min; Room temperature is centrifugal;
8) 75% ethanol washes one time, dries; Room temperature is centrifugal;
9) add stock solution V resuspended, 4 DEG C of dissolvings of spending the night, obtain anaerobic grain sludge microorganism total DNA.
Preferably, the thalline described in step 1) is centrifugal is, at 7000 ~ 9000r/min centrifugation, 3 ~ 8min; Preferred, at 8000r/min centrifugation 5min.
Preferably, the solids DNA stock solution I described in step 1) washes 2 times, and each usage quantity of DNA stock solution I is 5 ~ 10 times of thalline volume; Add DNA stock solution I described in step 1), add-on is 10 ~ 15 times of thalline volume.
Preferably, step 2) described in the add-on of N,O-Diacetylmuramidase be 0.05 ~ 0.2g/ml thalline; Preferred, the add-on of N,O-Diacetylmuramidase is 0.01g/ml thalline.
Preferably, step 2) described in the add-on of stock solution II be 30 ~ 80 μ L/ml thalline.Preferred, step 2) described in the add-on of stock solution II be 50 μ L/ml thalline.
Preferably, the add-on of the stock solution III described in step 3) is 70 ~ 120 μ L/ml thalline.Preferred, the add-on of the stock solution III described in step 3) is 100 μ L/ml thalline.
Preferably, the add-on of the stock solution IV described in step 4) is 60 ~ 100 μ L/ml thalline.Preferred, the add-on of the stock solution III described in step 4) is 80 μ L/ml thalline.
Preferably, in step 4), 65 DEG C of incubation 10min.
Preferably, in step 5), the add-on of phenol, chloroform and primary isoamyl alcohol mixed solution is 700 ~ 900 μ L/ml thalline; More preferably 800 μ L/ml thalline.Phenol: chloroform: primary isoamyl alcohol=22 ~ 28: 20 ~ 26: 1, volume ratio.Preferred, phenol: chloroform: primary isoamyl alcohol=25: 24: 1, volume ratio.
Preferably, in step 6), the add-on of chloroform and primary isoamyl alcohol mixed solution is 700 ~ 900 μ L/ml thalline; More preferably 800 μ L/ml thalline.Chloroform: primary isoamyl alcohol=20 ~ 26: 1, volume ratio.Preferred, chloroform: primary isoamyl alcohol=24: 1, volume ratio.
Preferably, step 5) and 6) described in centrifugal be 11000 ~ 13000r/min, 2 ~ 8 DEG C, centrifugal 5 ~ 15min.Preferred, step 5) and 6) described in centrifugal be 12000r/min, 4 DEG C, centrifugal 10min.
Preferably, the quantity of isopropanol described in step 7) is 700 ~ 900 μ L ,/ml thalline; More preferably, quantity of isopropanol is 800 μ L/ml thalline.Preferably, step 7) and 8) described in room temperature centrifugal be the centrifugal 5 ~ 15min of 12000 ~ 14000r/min.Preferred, the room temperature described in step 7) is centrifugal is the centrifugal 10min of 13000r/min; Step 7) and 8) described in room temperature centrifugal be 12000r/min, 5min.
Preferably, in step 9), the add-on of stock solution V is 30 μ L/ml thalline; Preferred, the add-on of stock solution V is 30 μ L/ml thalline.
Thalline of the present invention is black particle shape anaerobic activated sludge, and particle adds the EP pipe determination volume of 1.5ml.
1mL thalline is centrifugal, and 8000r/min, 5min, DNA commonly use stock solution I and wash 2 times.Add 565mL stock solution I, mixing.Add 0.1g N,O-Diacetylmuramidase, mixing, 37 DEG C of incubation 1h, add 50 μ L stock solutions II, mixing, 37 DEG C of incubation 1h.Add 100 μ L stock solutions III, mixing, adds 80 μ L stock solutions IV, mixing, 65 DEG C of incubation 10min.Add 800 μ L phenol: chloroform: primary isoamyl alcohol, soft 8 word mixings, 10min.Centrifugal 12000r/min, 4 DEG C, 10min.Get supernatant, add 800 μ L phenol: chloroform: primary isoamyl alcohol mixed solution (phenol: chloroform: primary isoamyl alcohol=25: 24: 1, volume ratio), soft 8 word mixings, 10min.Centrifugal 12000r/min, 4 DEG C, 10min.Get supernatant, add 800 μ L chloroforms: primary isoamyl alcohol mixed solution (chloroform: primary isoamyl alcohol=24: 1, volume ratio), soft 8 word mixings, 10min, centrifugal 12000r/min, 4 DEG C, 10min.Get supernatant, add 800 μ L Virahols, softly mix ,-20 DEG C, 30min.Room temperature centrifugal 13000r/min, 10min.75% ethanol washes one time, dries.Room temperature centrifugal 12000r/min, 5min.30 μ L stock solutions V are resuspended, 4 DEG C of dissolvings of spending the night, for subsequent use.
Extract DNA stock solution I: 1mmolEDTA+10mmolTris-Hcl and be mixed with 100ml solution; EDTA is ethylenediamine tetraacetic acid (EDTA); Tris-Hcl is three (methylol) aminomethane buffer solution;
Extract DNA stock solution II: 20mg/mL Proteinase K 20 μ L+10%SDS10ml and be mixed with 100ml solution; SDS is sodium laurylsulfonate;
Extract DNA stock solution III: 5molNaCl+0.1%CTAB10ml and be mixed with 100ml solution; CTAB is cetyl trimethylammonium bromide;
Extract DNA stock solution IV: 10%CTAB10ml+0.8molNaCl50ml and be mixed with 100ml solution;
Extract DNA stock solution V: 1mmolEDTA+10mmolTris-HCl+2mmolBSA and be mixed with 100ml solution; BSA is bovine serum albumin;
In the present invention, if do not have specified otherwise, solvent is water.The part that the present invention does not describe in detail, all can adopt prior art.
Beneficial effect of the present invention is:
1, the extracting solution that prepared by the present invention is N,O-Diacetylmuramidase, Proteinase K, SDS, is beneficial to microorganism wall cracking.
2, contain BSA in the extracting solution that prepared by the present invention, the stability that PCR reacts can be promoted.
3, the extract recipe that prepared by the present invention forms, and is the conventional reagent that laboratory generally adopts, good economy performance.
4, the extracting solution prepared of the present invention nontoxic, have no irritating odor, operational safety.
5, this invention simplifies microorganism total DNA extraction step, achieve the microorganism total DNA extracting anaerobic grain sludge fast and efficiently.
6, the extracting solution extraction effect prepared of the present invention is good, and the impurities left such as protein, phenols is less.
Accompanying drawing explanation
Fig. 1 is the macro genome DNA of embodiment 1;
Fig. 2 is the macro genome DNA of embodiment 2.
Embodiment
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
Active particle mud sample takes from Feitian, Yucheng majoy sewage work of pharmaceutcal corporation, Ltd.
1mL thalline is centrifugal, and 8000r/min, 5min, DNA commonly use stock solution I and wash 2 times.Add 565mL stock solution I, mixing.Add 0.1g N,O-Diacetylmuramidase, mixing, 37 DEG C of incubation 1h, add 50 μ L stock solutions II, mixing, 37 DEG C of incubation 1h.Add 100 μ L stock solutions III, mixing, adds 80 μ L stock solutions IV, mixing, 65 DEG C of incubation 10min.Get supernatant, add 800 μ L phenol: chloroform: primary isoamyl alcohol mixed solution (phenol: chloroform: primary isoamyl alcohol=25: 24: 1, volume ratio), soft 8 word mixings, 10min.Centrifugal 12000r/min, 4 DEG C, 10min.Get supernatant, add 800 μ L chloroforms: primary isoamyl alcohol mixed solution (chloroform: primary isoamyl alcohol=24: 1, volume ratio), soft 8 word mixings, 10min, centrifugal 12000r/min, 4 DEG C, 10min.Get supernatant, add 800 μ L Virahols, softly mix ,-20 DEG C, 30min.Room temperature centrifugal 13000r/min, 10min.75% ethanol washes one time, dries.Room temperature centrifugal 12000r/min, 5min.30 μ L stock solutions V are resuspended, 4 DEG C of dissolvings of spending the night.Get 1 μ L macro genome DNA, dilute 50 times, carry out quality examination with ultraviolet spectrophotometer.Get 5 μ L macro genome DNAs, detect DNA band with agarose gel electrophoresis.
STb gene size is about 21kb, and OD260/OD280 is 1.89.Its agarose electrophoresis detected result shows, and DNA band is more clear bright, sees Fig. 1.Detected result shows that DNA extraction total amount is high, purity is high.Use known detection method.
Embodiment 2
Active particle mud sample takes from sewage work of bowling Po Co., Ltd of Yucheng majoy.
1mL thalline is centrifugal, and 8000r/min, 5min, DNA commonly use stock solution I and wash 2 times.Add 565mL stock solution I, mixing.Add 0.1g N,O-Diacetylmuramidase, mixing, 37 DEG C of incubation 1h, add 50 μ L stock solutions II, mixing, 37 DEG C of incubation 1h.Add 100 μ L stock solutions III, mixing, adds 80 μ L stock solutions IV, mixing, 65 DEG C of incubation 10min.Add 800 μ L phenol: chloroform: primary isoamyl alcohol mixed solution (phenol: chloroform: primary isoamyl alcohol=25: 24: 1, volume ratio), soft 8 word mixings, 10min.Centrifugal 12000r/min, 4 DEG C, 10min.Get supernatant, add 800 μ L chloroforms: primary isoamyl alcohol mixed solution (chloroform: primary isoamyl alcohol=24: 1, volume ratio), soft 8 word mixings, 10min, centrifugal 12000r/min, 4 DEG C, 10min.Get supernatant, add 800 μ L Virahols, softly mix ,-20 DEG C, 30min.Room temperature centrifugal 13000r/min, 10min.75% ethanol washes one time, dries.Room temperature centrifugal 12000r/min, 5min.30 μ L stock solutions V are resuspended, 4 DEG C of dissolvings of spending the night.Get 1 μ L macro genome DNA, dilute 50 times, carry out quality examination with ultraviolet spectrophotometer.Get 5 μ L macro genome DNAs, detect DNA band with agarose gel electrophoresis.
STb gene size is about 21kb, and OD260/OD280 is 1.85.Its agarose electrophoresis detected result shows, and DNA band is more clear bright, sees Fig. 2.Detected result shows that DNA extraction total amount is high, purity is high.
Although above-mentioned, the specific embodiment of the present invention is described; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (13)
1. extract a method for anaerobic grain sludge microorganism total DNA, comprise the following steps:
1) thalline is centrifugal,
Measure the thalline of certain volume, put into centrifuge; Discard supernatant, bottom solids DNA stock solution I washes 2 times; Add DNA stock solution I, mixing; Described thalline is centrifugal is, at 7000 ~ 9000r/min centrifugation, 3 ~ 8min;
2) add N,O-Diacetylmuramidase, mixing, 30 ~ 40 DEG C of incubation 0.5 ~ 2h, add stock solution II, mixing, 30 ~ 40 DEG C of incubation 0.5 ~ 2h; Step 2) described in the add-on of N,O-Diacetylmuramidase be 0.05 ~ 0.2g/ml thalline; The add-on of described stock solution II is 30 ~ 80 μ L/ml thalline;
3) add stock solution III, mixing, step 3) described in the add-on of stock solution III be 70 ~ 120 μ L/ml thalline;
4) stock solution IV is added, mixing, 60-70 DEG C of incubation 5-15min;
5) phenol, chloroform and primary isoamyl alcohol mixed solution is added, soft 8 word mixing 5-15min; Room temperature leaves standstill 5-15min; Centrifugal;
6) get supernatant, add chloroform and primary isoamyl alcohol mixed solution, soft 8 word mixing 5-15min; Room temperature leaves standstill 5-15min; Centrifugal;
7) get supernatant, add Virahol, softly mix ,-20 DEG C, leave standstill 30min; Room temperature is centrifugal;
8) 75% ethanol washes one time, dries; Room temperature is centrifugal;
9) add stock solution V resuspended, 4 DEG C of dissolvings of spending the night, obtain anaerobic grain sludge microorganism total DNA;
Wherein, stock solution I: 1mmolEDTA+10mmolTris-HCl is mixed with 100ml solution;
Stock solution II: 20mg/mL Proteinase K 20 μ L+10%SDS10ml is mixed with 100ml solution;
Stock solution III: 5molNaCl+0.1%CTAB10ml is mixed with 100ml solution;
Stock solution IV: 10%CTAB10ml+0.8molNaCl50ml is mixed with 100ml solution;
Stock solution V: 1mmolEDTA+10mmolTris-HCl+2mmolBSA is mixed with 100ml solution.
2. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total DNA, is characterized in that, step 1) described in thalline be centrifugally, at 8000r/min centrifugation 5min;
Step 1) described in solids DNA stock solution I wash 2 times, each usage quantity of DNA stock solution I is 5 ~ 10 times of thalline volume; Step 1) described in add DNA stock solution I, add-on is 10 ~ 15 times of thalline volume.
3. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total DNA, is characterized in that, step 2) described in the add-on of stock solution II be 50 μ L/ml thalline.
4. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total DNA, is characterized in that, step 3) described in the add-on of stock solution III be 100 μ L/ml thalline.
5. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total DNA, is characterized in that, step 4) described in the add-on of stock solution IV be 60 ~ 100 μ L/ml thalline;
Step 4) in, 65 DEG C of incubation 10min.
6. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total DNA, is characterized in that, step 4) described in the add-on of stock solution III be 80 μ L/ml thalline.
7. the method extracting anaerobic grain sludge microorganism total DNA as claimed in claim 1, is characterized in that, step 5) in, the add-on of phenol, chloroform and primary isoamyl alcohol mixed solution is 700 ~ 900 μ L/ml thalline; Phenol: chloroform: primary isoamyl alcohol=22 ~ 28: 20 ~ 26: 1, volume ratio;
Step 6) in, the add-on of chloroform and primary isoamyl alcohol mixed solution is 700 ~ 900 μ L/ml thalline; Chloroform: primary isoamyl alcohol=20 ~ 26: 1, volume ratio.
8. the method extracting anaerobic grain sludge microorganism total DNA as claimed in claim 1, is characterized in that, step 5) in, phenol: chloroform: primary isoamyl alcohol=25: 24: 1, volume ratio; Step 6) in, chloroform: primary isoamyl alcohol=24: 1, volume ratio.
9. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total DNA, is characterized in that, step 5) and 6) described in centrifugal be 11000 ~ 13000r/min, 2 ~ 8 DEG C, centrifugal 5 ~ 15min.
10. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total DNA, is characterized in that, step 7) described in quantity of isopropanol be 700 ~ 900 μ L ,/ml thalline; Step 7) and 8) described in room temperature centrifugal be the centrifugal 5 ~ 15min of 12000 ~ 14000r/min.
11. methods extracting as claimed in claim 1 anaerobic grain sludge microorganism total DNAs, is characterized in that, step 7) in quantity of isopropanol be 800 μ L/ml thalline; Step 7) described in room temperature centrifugal be the centrifugal 10min of 13000r/min; Step 8) described in room temperature centrifugal be 12000r/min, 5min.
12. methods extracting as claimed in claim 1 anaerobic grain sludge microorganism total DNAs, is characterized in that, step 9) in the add-on of stock solution V be 30 μ L/ml thalline.
13. methods extracting as claimed in claim 1 anaerobic grain sludge microorganism total DNAs, is characterized in that, step 9) in the add-on of stock solution V be 30 μ L/ml thalline.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161987A (en) * | 2010-02-20 | 2011-08-24 | 大连水产学院 | Method for extracting genomic deoxyribonucleic aid (DNA) of sediment microorganisms in mariculture pond |
| CN102220309A (en) * | 2011-04-11 | 2011-10-19 | 吉林建筑工程学院 | Method for extracting DNA (deoxyribonucleic acid) of active sludge in anaerobic reactor |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161987A (en) * | 2010-02-20 | 2011-08-24 | 大连水产学院 | Method for extracting genomic deoxyribonucleic aid (DNA) of sediment microorganisms in mariculture pond |
| CN102220309A (en) * | 2011-04-11 | 2011-10-19 | 吉林建筑工程学院 | Method for extracting DNA (deoxyribonucleic acid) of active sludge in anaerobic reactor |
Non-Patent Citations (3)
| Title |
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| 东北设施黑土土壤微生物总DNA 提取方法探讨;刘昕昕等;《生物技术》;20130430;第23卷(第2期);第46页1.2.1.1-1.2.1.2节 * |
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