CN102382818A - Method for quickly and efficiently extracting total DNA in drinking water - Google Patents
Method for quickly and efficiently extracting total DNA in drinking water Download PDFInfo
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Abstract
The invention discloses a method for quickly and efficiently extracting total DNA in drinking water, and belongs to the technical field of extraction and purification of total DNA in drinking water. The method is used for solving the problems that the drinking water has extremely low microbial biomass, the total DNA of effective concentration is difficulty extracted by the conventional filtration and concentration method and complex environmental media affect the purity of the extracted DNA. The method comprises the following steps of: filtering microbes in the drinking water through a water purification device; eluting the microbes from the filtering material by adopting ultrasonic; filtering the eluent by using a filter membrane; extracting the total DNA of the microbes on the filter membrane directly by combining physical grinding and chemical cracking; and washing the extracted total DNA by using absolute ethanol, and dissolving the dried total DNA into ultrapure water or a buffer solution. The method is economic, convenient and stable; the extracted DNA has relatively high concentration and purity; and a new method is provided for application of an environmental molecular biology technology.
Description
Technical field
The present invention is that a kind of to extract the series methods of total DNA from tap water integrated, says so more specifically and extracts the method for rapidly and efficiently extracting total DNA total DNA from tap water.
Background technology
Drinking water safety and human health are closely bound up, and structure of community, the behavior of mikrobe return the relation inseparable with having of numerous diseases in the research report tap water, and its health risk can not be ignored.And the research to mikrobe the tap water is one of numerous important analysis means from total dna level.
Tap water living weight after the sterilization is extremely low, and traditional filtering and concentrating mode is difficult to extract total DNA of effective concentration.In research in the past, the investigator usually obtains total DNA in the tap water with other indirect modes, as gathering tap water storage pool, the microbial film (Hong in pipeline, water tap, the water meter; P.Y., Hwang, C.C., Ling; F.Q., Andersen, G.L., LeChevallier; M.W., and Liu, W.T. 2010. Pyrosequencing analysis of bacterial biofilm communities in water meters of a drinking water distribution system. Appl. Environ. Microbiol. 76,5631-5635.); Perhaps utilize homemade microbial film collection device to obtain microbial film (Schwartz, T., Kohnen; W., Jansen, B.; Obst, U., 2003. Detection of antibiotic-resistant bacteria and their resistance genes in wastewater; Surface water, and drinking water biofilms. Fems. Microbiol. Ecol. 43,325-335.).These methods can be obtained the total DNA in the environmental sample really, but also have a series of problems simultaneously.
Its biological community structure in the microbial film forming process, functional form inevitably will change (Stoodley, P.; Sauer, K., Davies; D.G., Costerton, J.W.; 2002. Biofilms as complex differentiated communities. Annu. Rev. Microbiol. 56,187-209.), it is not enough to reflect definitely the truth of mikrobe in the tap water.The microbial film formation time is grown (Kalmbach simultaneously; S.; Manz, W., Szewzyk; U.; 1997. Dynamics of biofilm formation in drinking water:Phylogenetic affiliation and metabolic potential of single cells assessed by formazan reduction and in situ hybridization. Fems. Microbiol. Ecol. 22 265-279.), is difficult to obtain fast at short notice total DNA sample.
Summary of the invention
1. invent the technical problem that will solve
To can't directly from tap water, extracting total DNA now; Perhaps process for extracting exists speed slow; The situation of poor accuracy, the present invention provides a kind of method of rapidly and efficiently extracting total DNA in the tap water, adopts serial of methods to solve the problem from the sample collecting to the total DNA extraction; Can improve the speed and the efficient of total DNA extraction, for follow-up on total DNA water body, research laid a good foundation.
2. technical scheme of the present invention
Concrete enforcement schemes of the present invention and simple principle are following:
A kind of method of rapidly and efficiently extracting total DNA in the tap water the steps include:
The first step: sampling and sample pre-treatments.
At first choose suitable sampling point, require effluent quality up to standard, no obvious particulate contamination in the water, hydraulic pressure is normal.Open the thief hole water tap, normal fast sky was put 5 minutes, removed the stagnant water that is detained in the water pipe, used spirit lamp calcination water tap then, killed near the bacterium of water tap.
A kind of family type water purifier is installed on the water tap of sterilization, and (model: Torayvino MK2-EG), installation method specifically can be with reference to the specification sheets of this water purifier according to dissimilar water taps and difference.After the installation, the tap of fetching boiling water lets water from the filter core water outlet water outlet of this water purifier, and the drainage that picks up counting is thoroughly blocked the rear until this filter core and taken off, and is generally 24-36 hour according to the different drainage times of hydraulic pressure, and the filtered water total amount is at the 1500-2000 liter.And directly use conventional vacuum pump and 0.45 micron membrane filtration maximum amount of water 8-10 liter (look the water quality situation and different), this kind drainage mode has greatly improved the speed and the volume of drainage.
In time water purifier cuts out after filter accomplishing and take off, unload lower filter, the filter core front end is checked that the window position opens with hacksaw; Take out the inside multiwalled hollow fiber membrane; Be placed in 500 ml beakers of sterilization, add 200 milliliters of ultrapure waters, light shaking lets hollow fiber membrane evenly be scattered in the flask.Then beaker is positioned over 20 minutes (models: KuDos HK3310HP of sonic oscillation in the Ultrasonic Cleaners; Frequency: 53Khz), per 100 ml waters were descended 0.45 micron cellulose acetate filter membrane at 0.98 MPa normal atmosphere after ultrasonic, filter membrane is placed 4 degree refrigerators and is given over to next step use.
Second step: total DNA extraction in the tap water.
That the extraction of total DNA is adopted in the tap water is the FastDNA ﹫ SPIN Kit for Soil of MP Biomedicals company.At first filter membrane is shredded as far as possible, fragment is encased in 2 milliliters sample dissociation pipe, do following processing, the equal Eppendorf centrifuge 5424 of whizzer model that this step adopts according to the operational guidance of this test kit.
(1) in the sample dissociation pipe, adds 978 microlitre phosphate buffered saline buffers and 122 microlitre MT damping fluids.
(2) with grinding bead homogenizer (model: cracking 90 seconds under 4800 rev/mins of conditions BioSpec Beadbeater-1), thoroughly disruption of microorganisms cell.
(3) with sample dissociation pipe under 14000 g rotating speeds centrifugal 15 minutes.
(4) supernatant in the pipe of centrifugal back is transferred in 2.0 milliliters of new centrifuge tubes, added 250 microlitre albumen precipitation solution, fully vibrated 2 minutes.
(5) recentrifuge was removed deposition in 5 minutes under the 14000g, and supernatant is all transferred in 15 milliliters of clean centrifuge tubes.
(6) with a kind of can combine dna solution vibration to shake up after, add in 1 milliliter of this solution to 15 milliliter centrifuge tube.
(7) after 2 minutes centrifuge tube is placed on the support static 3 minutes with vortex vibrator vibration centrifuge tube, lets silica dioxide granule deposition in the solution.
(8) remove 500 microlitre supernatants.
(9) with mixture remaining in centrifuge tube mixing again, get 600 microlitre solution to the centrifuging post, 14000g removes the liquid in the collection tube after centrifugal 1 minute.Repeat said process, continue to join on the Filter column remaining mixture centrifugal.
(10) the SEWS-M solution that adds 500 microlitres is blown and beaten solution with liquid-transfering gun gently in centrifugal post, with the thorough mixing of the inside mixture.
(11) 14000g is centrifugal 1 minute, abandons liquid in the collection tube.
(12) do not add any reagent, centrifugal 2 minutes of 14000g removes ethanol residual in the centrifugal post, abandons former collection tube, changes new 1.5 milliliters of clean centrifuge tubes.
(13) with centrifugal post dry air 5 minutes at room temperature.
(14) sterilized water of 50 microlitres of adding earlier, 55 degree preheatings stirs with liquid-transfering gun in centrifugal post gently, if dissolving not exclusively, then stirs at adding 10 microlitre sterilized waters, and silicon-dioxide all becomes molten state in centrifugal post.
(15) with heating in the centrifugal post placement 55 degree baking ovens 5 minutes.
(16) 14000g collects to such an extent that liquid promptly extracts total DNA in the gained tap water after centrifugal 1 minute in the centrifuge tube, and subzero 20 degree are preserved.
After accomplishing above-mentioned two steps, can carry out total DNA concentration and purity and identify.
Total DNA concentration and purity are identified and can be adopted spectrophotometry and agarose electrophoresis.Concrete grammar and step are following:
Spectrophotometry
Absorbance under 260 nano wave lengths is the concentration of total DNA quantitatively, and A260/A280 indicates the purity of total DNA.The present invention uses in the spectrophotometry tap water total DNA concentration and the used instrument of the purity multi-functional ELIASA (model: Synergy H4) as Bio-tek company.Operation steps is following:
(1) opens computer and multi-functional ELIASA preheating 10 minutes.
(2) add 2 microlitre sterilized waters to Take3 ultramicron check-out console, as negative control.Get the total DNA sample of 2 microlitres then and be added to other above hole, to be determined.
(3) open multi-functional ELIASA software Gen5 (version number: 1.10.8), measure total DNA concentration and purity in the tap water.
Agarose electrophoresis
Agarose gel electrophoresis be separate being used to of using always, the method for identification of dna, RNA molecule mixture; This electrophoresis method with agar gel as upholder; Utilize electrocharge effect and the molecular sieve effect of dna molecular when swimming; Reach the purpose of separating mixture, and can carry out qualitative and relative quantitative assay to extracting gained DNA sample according to the deposition condition situation, the concrete operations step is following:
(1) takes by weighing 0.4 an amount of gram agarose powder, be put in the Erlenmeyer flask, add 40 milliliters of an amount of 1 * TBE electrophoretic buffers.Be heated to fully then and dissolve, solution is transparent.Shake up slightly, get glue.Be cooled to about 60 ℃, in glue, adding 2 an amount of microlitre ethidium bromide to concentration is 0.5 mcg/ml.
(2) horizontal positioned glue groove is at one end inserted good comb, in groove, slowly pours the glue that has been chilled to about 60 degree into, makes it to form the glue face of even level.
(3) treat that gelling is solid after, carefully pull up comb, make the application of sample nose end put the negative electrode section and put in the electrophoresis chamber.
(4) in groove, add 1 * TBE electrophoretic buffer, covered the glue face to liquid level.
(5), total DNA sample adding is gone up in the appearance hole with liquid-transfering gun with total DNA sample mixing sample preparation of 1 microlitre Loading Buffer (0.25% tetrabromophenol sulfonphthalein and aseptic sucrose) with 5 microlitres.Establish DNA marker and negative control swimming lane simultaneously.
(6) connect electrophoresis apparatus and electrophoresis chamber, and energized, regulate voltage stabilizing and be output as 120 volts, electrophoresis time 20 minutes.
(7) gel is placed on the sample table of gel imaging appearance the bubble tiling of rushing.Shut the sample chamber external door, open uv lamp (wavelength 254 nanometers), observe through the computer shooting.
According to above two kinds of methods is the situation such as concentration, purity and integrity of the total DNA in the tap water that extracts of decidable.
3. beneficial effect
The invention provides a kind of method of extracting total DNA in the tap water, rapidly and efficiently is the most significant advantage of the inventive method, finishes to extracting evaluation from sample collecting, and the time is 2-3 days altogether.And described in the background technology document, microbial film forms minimum needs 30 days in traditional tap water.Compare former process for extracting, total DNA concentration that the inventive method extracts is high, and purity is good, has good applicability.
Description of drawings
Fig. 1 is a Torayvino MK2-EG water purifier filtration experiment chamber tap water synoptic diagram;
Fig. 2 filters northern river mouth water factory sterilization tap water synoptic diagram after 5 minutes for Torayvino MK2-EG water purifier;
Fig. 3 is a circulation ability of swimming vacuum pump suction filtration setting drawing;
The total DNA electrophorogram of tap water that Fig. 4 obtains for embodiment 1 and embodiment 2.
Fig. 5 is the 16S rDNA fragment electrophorogram of total DNA in the pcr amplification tap water.
Embodiment
In order better to set forth step and the effect that the inventive method is extracted total DNA in the tap water, further specify the present invention in conjunction with specific embodiment, specific as follows:
Embodiment 1
The first step: sampling and sample pre-treatments
(1) this instance has selected the laboratory tap water as sampling spot, requires effluent quality up to standard, no obvious particulate contamination in the water, and hydraulic pressure is normal.Open the thief hole water tap, normal fast sky was put 5 minutes, removed the stagnant water that is detained in the water pipe, used spirit lamp calcination water tap then, killed near the bacterium of water tap; A kind of family type water purifier Torayvino MK2-EG is installed on the foam encasement type water tap of sterilization, shown in this water purifier installation guide, selects Type B external spiral foam encasement.At first taking off from main frame and install with nut and set collar, take off the foam encasement of water tap, will install with nut and pass water tap, with spanner foam encasement vertically is screwed into water tap, tighten at last to install and use nut, is installation behind the fixed host computer.The tap of fetching boiling water then lets water from the filter core water outlet water outlet of this water purifier, and the drainage that picks up counting is thoroughly blocked the rear until this filter core and taken off, and is as shown in Figure 1:
(2) filter 23 took off the hollow fiber membrane in the water purifier after 6 hours, was placed in 500 ml beakers of sterilization, added 200 milliliters of ultrapure waters, and light shaking lets hollow fiber membrane evenly be scattered in the flask.Then beaker is positioned over 20 minutes (models: KuDos HK3310HP of sonic oscillation in the Ultrasonic Cleaners; Frequency: 53Khz), per 100 ml waters were descended 0.45 micron cellulose acetate filter membrane at 0.98 MPa normal atmosphere after ultrasonic, filter membrane is placed 4 degree refrigerators and is given over to next step use.As shown in Figure 2:
Second step: total DNA extraction in the tap water.
That the extraction of total DNA is adopted in the tap water is the FastDNA ﹫ SPIN Kit for Soil of MP Biomedicals company.At first filter membrane is shredded as far as possible, fragment is encased in 2 milliliters sample dissociation pipe, do following processing, the equal Eppendorf centrifuge 5424 of whizzer model that this step adopts according to the operational guidance of this test kit.
(1) in the sample dissociation pipe, adds 978 microlitre phosphate buffered saline buffers and 122 microlitre MT damping fluids.
(2) with grinding bead homogenizer (model: cracking 90 seconds under 4800 rev/mins of conditions BioSpec Beadbeater-1), thoroughly disruption of microorganisms cell.
(3) with sample dissociation pipe under 14000 g rotating speeds centrifugal 15 minutes
(4) supernatant in the pipe of centrifugal back is transferred in 2.0 milliliters of new centrifuge tubes, added 250 microlitre albumen precipitation solution, fully vibrated 2 minutes.
(5) recentrifuge was removed deposition in 5 minutes under the 14000g, and supernatant is all transferred in 15 milliliters of clean centrifuge tubes.
(6) with a kind of can combine dna solution vibration to shake up after, add in 1 milliliter of this solution to 15 milliliter centrifuge tube.
(7) after 2 minutes centrifuge tube is placed on the support static 3 minutes with vortex vibrator vibration centrifuge tube, lets silica dioxide granule deposition in the solution.
(8) remove 500 microlitre supernatants.
(9) with mixture remaining in centrifuge tube mixing again, get 600 microlitre solution to the centrifuging post, 14000g removes the liquid in the collection tube after centrifugal 1 minute.Repeat said process, continue to join on the Filter column remaining mixture centrifugal.
(10) the SEWS-M solution that adds 500 microlitres is blown and beaten solution with liquid-transfering gun gently in centrifugal post, with the thorough mixing of the inside mixture.
(11) 14000g is centrifugal 1 minute, abandons liquid in the collection tube.
(12) do not add any reagent, centrifugal 2 minutes of 14000g removes ethanol residual in the centrifugal post, abandons former collection tube, changes new 1.5 milliliters of clean centrifuge tubes.
(13) with centrifugal post dry air 5 minutes at room temperature.
(14) sterilized water of 50 microlitres of adding earlier, 55 degree preheatings stirs with liquid-transfering gun in centrifugal post gently, if dissolving not exclusively, then stirs at adding 10 microlitre sterilized waters, and silicon-dioxide all becomes molten state in centrifugal post.
(15) with heating in the centrifugal post placement 55 degree baking ovens 5 minutes.
(16) 14000g collects to such an extent that liquid promptly extracts total DNA in the gained tap water after centrifugal 1 minute in the centrifuge tube, and subzero 20 degree are preserved.
The 3rd step: total DNA concentration and purity are identified.
Adopt agarose electrophoresis.Concrete grammar and step are following:
(1) takes by weighing 0.4 an amount of gram agarose powder, be put in the Erlenmeyer flask, add 40 milliliters of an amount of 1 * TBE electrophoretic buffers.Be heated to fully then and dissolve, solution is transparent.Shake up slightly, get glue.Be cooled to about 60 ℃, in glue, adding 2 an amount of microlitre ethidium bromide to concentration is 0.5 mcg/ml.
(2) horizontal positioned glue groove is at one end inserted good comb, in groove, slowly pours the glue that has been chilled to about 60 degree into, makes it to form the glue face of even level.
(3) treat that gelling is solid after, carefully pull up comb, make the application of sample nose end put the negative electrode section and put in the electrophoresis chamber.
(4) in groove, add 1 * TBE electrophoretic buffer, covered the glue face to liquid level.
(5), total DNA sample adding is gone up in the appearance hole with liquid-transfering gun with total DNA sample mixing sample preparation of 1 microlitre Loading Buffer (0.25% tetrabromophenol sulfonphthalein and aseptic sucrose) with 5 microlitres.Establish DNA marker and negative control swimming lane simultaneously.
(6) connect electrophoresis apparatus and electrophoresis chamber, and energized, regulate voltage stabilizing and be output as 120 volts, electrophoresis time 20 minutes.
(7) gel is placed on the sample table of gel imaging appearance the bubble tiling of rushing.Shut the sample chamber external door, open uv lamp (wavelength 254 nanometers), observe through the computer shooting.
With agarose electrophoresis qualitative detection extraction effect, electrophorogram is as shown in Figure 3, and wherein standard substance DL2000 and λ-Hind III all is TaKaRa Company products, and DW1 and DW2 be total DNA gained for the laboratory water discharge of water faucet extracts.
Synergy H4 ELIASA is measured total DNA concentration and purity through Bio-tek company, and concrete determination data is as shown in table 1 below:
Table 1: total DNA concentration and purity in the tap water
| Sample reads | 260 Raw | 280 Raw | 320 Raw | 260 | 280 | 260/280 | ng/ul |
| DW1 | 0.463 | 0.277 | 0.050 | 0.437 | 0.237 | 1.848 | 437.271 |
| DW2 | 0.605 | 0.37 | 0.083 | 0.546 | 0.297 | 1.840 | 546.169 |
Annotate: wherein first classify sample number into spectrum as, second to the 4th row each sample of position absorbancy raw value under different wave length, the 5th to the 7th row are individually absorbance and the ratio between the two after the background correction value, and the 8th classifies total DNA ultimate density value as.
Can know by data in the table; Use the inventive method can effectively extract total DNA in the tap water of higher concentration; Its concentration is all in that 400 nanograms/more than the microlitre, absorbance ratio proves that it is not polluted by protein or RNA under 260 nanometers and 280 nanometers between 1.8 and 2.0; Purity is higher, can on this total DNA basis, carry out each item downstream molecules biologic operation.
Embodiment 2
The first step: sampling
(1) this instance has selected waterworks, river mouth, north, Nanjing to sterilize back 5 minutes water outlets as sampling spot; Open the thief hole water tap, normal fast sky was put 5 minutes, removed the stagnant water that is detained in the water pipe; Use spirit lamp calcination water tap then, kill near the bacterium of water tap; A kind of family type water purifier Torayvino MK2-EG is installed on the no wheel rim type water tap of sterilization, shown in this water purifier installation guide, selects D type mounting block.At first take off and install, with the bolted fastener main frame of all packing into, gently about the about half cycle of rotation screw mounting block with nut and set collar from main frame; Temporary transient fixing; But must reserve the gap, from the below main frame is forced into water tap, be installation behind the primary screw around equalization is tightened simultaneously.The tap of fetching boiling water then lets water from the filter core water outlet water outlet of this water purifier, and the drainage that picks up counting is thoroughly blocked the rear until this filter core and taken off, and is as shown in Figure 2:
Second step: total DNA extraction in the tap water (identical) with instance 1 step
The 3rd step: total DNA concentration and purity are identified (identical with instance 1 step)
Extract shown in the total DNA electrophorogram of gained like Fig. 3, DW3 and DW4 by northern river mouth water factory sterilize water outlet in back 5 minutes extracted total DNA, its band is clear to be concentrated, extraction effect is better.
Synergy H4 ELIASA is measured total DNA concentration and purity through Bio-tek company, and concrete determination data is as shown in table 2:
Table 2: total DNA concentration and purity in the tap water
| Sample reads | 260 Raw | 280 Raw | 320 Raw | 260 | 280 | 260/280 | ng/ul |
| DW3 | 0.321 | 0.202 | 0.061 | 0.273 | 0.145 | 1.882 | 272.659 |
| DW4 | 0.235 | 0.153 | 0.057 | 0.186 | 0.096 | 1.933 | 185.585 |
Annotate: wherein first classify sample number into spectrum as, second to the 4th row each sample of position absorbancy raw value under different wave length, the 5th to the 7th row are individually absorbance and the ratio between the two after the background correction value, and the 8th classifies total DNA ultimate density value as.
The living weight in the water outlet in back 5 minutes of sterilizing is obtained tap water than in the laboratory living weight is lower; Therefore show not 185.585 and 272.659 nanograms/microlitre through ELIASA concentration determination result; Though do not have total DNA concentration of laboratory water outlet high, this concentration also is enough to carry out any molecular biological downstream analysis.
Embodiment 3
Total DNA reflects template as PCR in this exemplary application embodiment 1 and the embodiment 2 extraction gained tap water, amplification 16S rDNA gene, and the total effect of DNA when carrying out the downstream molecules biologic operation in the inventive method extraction gained tap water used in check.
(1) 16S rDNA primer sequence
F:?AGAGTTTGATCCTGGCTCAG
R:?GGTTACCTTGTTACGACTT
(2) PCR reaction system
This reaction system TV is 30 microlitres, and wherein various strength of solution are following:
1. 10 * Buffer:3 microlitre
2. the dNTP:200 micromole is every liter
3. MgCl
2: every liter of 1.66 mmole
4. the Primers:0.166 mmole is every liter
⑤ Taq?polymerase：2?U
6. Template:100 nanogram
7. ddH
2The O:19.2 microlitre
(3) cycling condition
1. 94 degree are 5 minutes
2. 94 degree are 45 seconds
3. 56 degree are 45 seconds
4. 72 degree are 90 seconds
5. repeat 2. to arrive 4. 35 times
6. 94 degree are 8 minutes
This PCR reaction institute uses the pcr amplification appearance (model: Mycycler) of instrument as eppendorf company; The amplified fragments size is 1465 bp; PCR product electrophoresis step is identical with agarose electrophoresis step among the embodiment 1; Expanding effect is as shown in Figure 5; No matter can be found out by band among the figure, be water tap or the total DNA that extracts from the back 5 minutes water tap with two outlet of sterilizing can both amplify 16S rDNA condition well from the laboratory, proves that total DNA that this inventive method extracts can carry out the downstream molecules biologically.
Table 3: the english abbreviation and the implication thereof of the present invention's design
| Dummy suffix notation | English full name | The Chinese implication |
| DNA | Deoxyribonucleic acid | Deoxyribonucleotide |
| RNA | Ribonucleic acid | Ribonucleotide |
| PCR | Polymerase chain reaction | The polymerase chain reaction |
| dNTP | Deoxynucleoside triphosphates | 3-phosphoric acid thymus nucleic acid |
| U | Unit | The activity unit of enzyme |
Claims (3)
1. method of rapidly and efficiently extracting in the tap water total DNA, its step does
The first step: sampling and sample pre-treatments
On the water tap of sterilization, water purifier is installed, the tap of fetching boiling water lets the tap water of water quality reaching standard from the filter core water outlet water outlet of water purifier, and the drainage that picks up counting is thoroughly blocked the rear until this filter core and taken off;
In time water purifier cuts out after filtration is accomplished and take off, unload lower filter, take out the inside multiwalled hollow fiber membrane, be placed in the beaker of sterilization, add ultrapure water, light shaking lets hollow fiber membrane evenly be scattered in the flask;
Then beaker is positioned over sonic oscillation in the Ultrasonic Cleaners, water was descended 0.45 micron cellulose acetate filter membrane at 0.98 MPa normal atmosphere after ultrasonic;
Second step: total DNA extraction in the tap water
Filter membrane is shredded as far as possible, fragment is encased in the sample dissociation pipe, handle as follows:
(1) in the sample dissociation pipe, adds 978 microlitre phosphate buffered saline buffers and 122 microlitre MT damping fluids;
(2) with the cracking 90 seconds under 4800 rev/mins of conditions of grinding bead homogenizer, thoroughly disruption of microorganisms cell;
(3) with sample dissociation pipe under 14000 g rotating speeds centrifugal 15 minutes;
(4) supernatant in the pipe of centrifugal back is transferred in 2.0 milliliters of new centrifuge tubes, added 250 microlitre albumen precipitation solution, fully vibrated 2 minutes;
(5) recentrifuge was removed deposition in 5 minutes under the 14000g, and supernatant is all transferred in 15 milliliters of clean centrifuge tubes;
(6) with a kind of can combine dna solution vibration to shake up after, add in 1 milliliter of this solution to 15 milliliter centrifuge tube;
(7) after 2 minutes centrifuge tube is placed on the support static 3 minutes with vortex vibrator vibration centrifuge tube, lets silica dioxide granule deposition in the solution;
(8) remove 500 microlitre supernatants;
(9) with mixture remaining in centrifuge tube mixing again, get 600 microlitre solution to the centrifuging post, 14000g removes the liquid in the collection tube after centrifugal 1 minute; Repeat said process, continue to join on the Filter column remaining mixture centrifugal;
(10) the SEWS-M solution that adds 500 microlitres is blown and beaten solution with liquid-transfering gun gently in centrifugal post, with the thorough mixing of the inside mixture;
(11) 14000g is centrifugal 1 minute, abandons liquid in the collection tube;
(12) do not add any reagent, centrifugal 2 minutes of 14000g removes ethanol residual in the centrifugal post, abandons former collection tube, changes new 1.5 milliliters of clean centrifuge tubes;
(13) with centrifugal post dry air 5 minutes at room temperature;
(14) sterilized water of 50 microlitres of adding earlier, 55 degree preheatings stirs with liquid-transfering gun in centrifugal post gently, if dissolving not exclusively, then stirs at adding 10 microlitre sterilized waters, and silicon-dioxide all becomes molten state in centrifugal post;
(15) with heating in the centrifugal post placement 55 degree baking ovens 5 minutes;
(16) 14000g collects to such an extent that liquid promptly extracts total DNA in the gained tap water after centrifugal 1 minute in the centrifuge tube, and subzero 20 degree are preserved.
2. the method for rapidly and efficiently extracting total DNA in the tap water according to claim 1 is characterized in that at first choosing suitable sampling point in the first step sampling and the sample pre-treatments, requires effluent quality up to standard, no obvious particulate contamination in the water, and hydraulic pressure is normal; Open the thief hole water tap, normal fast sky was put 5 minutes, removed the stagnant water that is detained in the water pipe, used spirit lamp calcination water tap then, killed near the bacterium of water tap.
3. the method for rapidly and efficiently extracting total DNA in the tap water according to claim 1 is characterized in that a kind of family type water purifier Torayvino MK2-EG is installed on the water tap of sterilization in the first step sampling and the sample pre-treatments, after the installation; The tap of fetching boiling water; Let water from the filter core water outlet water outlet of this water purifier, the drainage that picks up counting is thoroughly blocked the rear until this filter core and is taken off; Be generally 24-36 hour according to the different drainage times of hydraulic pressure, the filtered water total amount is at the 1500-2000 liter.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102690806A (en) * | 2012-05-02 | 2012-09-26 | 江苏大学 | Method for simply and rapidly extracting total RNA from switchgrass tissue |
| CN104568680A (en) * | 2015-01-14 | 2015-04-29 | 浙江大学 | Community monitoring method of microorganism carried by air particulate matters with multiple particle sizes |
| CN111394348A (en) * | 2020-03-30 | 2020-07-10 | 南华大学 | Method for extracting and detecting free DNA in sewage |
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| CN1563422A (en) * | 2004-04-14 | 2005-01-12 | 重庆大学 | Method for fast detecting colibacillus in drinking water |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102690806A (en) * | 2012-05-02 | 2012-09-26 | 江苏大学 | Method for simply and rapidly extracting total RNA from switchgrass tissue |
| CN104568680A (en) * | 2015-01-14 | 2015-04-29 | 浙江大学 | Community monitoring method of microorganism carried by air particulate matters with multiple particle sizes |
| CN111394348A (en) * | 2020-03-30 | 2020-07-10 | 南华大学 | Method for extracting and detecting free DNA in sewage |
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