CN104560952A - Method for rapidly extracting DNA of gram-positive bacterium genome based on paramagnetic particle method - Google Patents
Method for rapidly extracting DNA of gram-positive bacterium genome based on paramagnetic particle method Download PDFInfo
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Abstract
The invention discloses a method for rapidly extracting DNA of gram-positive bacterium genome based on a paramagnetic particle method. The method comprises the following three steps: breaking gram-positive bacterium cells, freeing DNA, extracting impurity protein, and rapidly purifying DNA of the genome by using the paramagnetic particle method. Compared with a conventional method for rapidly extracting the DNA of the gram-positive bacterium genome, the method is capable of not only saving a great deal of extraction time, but also simplifying the experiment steps, and the DNA of the gram-positive bacterium genome can be relatively effectively, rapidly and economically extracted.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method based on paramagnetic particle method rapid extraction gram-positive microorganism genomic dna.
Background technology
Gram-positive microorganism can dye bacterium that is dark blue or purple with gramstaining, and Gram-negative bacteria can not be colored (but can paint red with contrast).Containing relatively large peptidoglycan in gram-positive bacteria cell wall, but often lack the second tunic and lipopolysaccharides layer that Gram-negative bacteria has.The cell walls of gram positive bacterium is thicker (20 ~ 80nm), and be mainly made up of peptidoglycan and teichoic acid, only have a Rotating fields, main component is peptidoglycan, accounts for 50% ~ 90% of cell walls dry weight.In dyeing course, when using Ethanol Treatment, cause the aperture in reticulated structure to diminish due to dehydration, permeability reduces, and makes Viola crystallina-Surgidine be retained in cell and not easily decolour, therefore, presents bluish voilet
Now, people start to carry out the research of Fast Detection Technique and the research of various biological property to gram-positive microorganism, current research mainly concentrates on the aspects such as virulence regulatory mechanism, superantigen mechanism of action, resistance, mycoderm formation and infection, but bases of these researchs are the extractions carrying out genomic dna.Because its cell walls of gram-positive microorganism is thicker, extracting genome DNA difficulty is comparatively large, time-consuming longer.
Effective extraction gram-positive microorganism genomic dna, can for carrying out genetic analysis and gene clone provides important guarantee, thus the research contents of redundant gene group.Therefore, develop a kind of gram-positive microorganism extracting genome DNA that is applicable to, convenient, fast, practical method, solve and use ordinary method to extract the problem that needed for gram-positive microorganism genome DNA sample, extraction time is long, significant.
Summary of the invention
The object of the present invention is to provide a kind of convenient, fast, practical method based on paramagnetic particle method rapid extraction gram-positive microorganism genomic dna, solve and use ordinary method to extract the problem that needed for gram-positive microorganism genomic dna, extraction time is long.
For achieving the above object, the present invention adopts following technical scheme:
Based on a method for paramagnetic particle method rapid extraction gram-positive microorganism genomic dna, comprise the fragmentation of gram-positive bacteria cell, dissociating of DNA and extracting of foreign protein, paramagnetic particle method fast purifying genomic dna, described method specifically comprises the following steps:
1) fragmentation of gram-positive bacteria cell: get 3ml gram-positive microorganism incubated overnight liquid in centrifuge tube, supernatant is abandoned after the centrifugal 1-5min of 10000-13000rpm/min, 0.2-0.5ml distilled water suspension precipitation is added in bacterial sediment, and add 50-80 μ l working fluid A, in 40 DEG C of insulation 10-50min, then add 30-60 μ l working fluid B and 5-10 μ l working fluid C, in 37 DEG C of insulation 30-60min after mixing, then add 0.4-0.6ml working fluid D, mix;
2) the extracting of free and foreign protein of DNA: in step 1) add 200-600 μ l working fluid E extracting in the mixed solution that obtains, then the centrifugal 10-30min of 10000-13000rpm/min, get a supernatant liquor in the 1.5ml centrifuge tube of sterilizing, add 200-600 μ l working fluid F extracting, then the centrifugal 10-30min of 10000-13000rpm/min, gets secondary supernatant liquor in aseptic 1.5ml centrifuge tube;
3) paramagnetic particle method fast purifying genomic dna: add with secondary supernatant liquor isopyknic working fluid G in the centrifuge tube containing secondary supernatant liquor, after concussion 10-30s, room temperature places 1-10min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, retain magnetic bead, then toward containing adding 0.5-1.5ml working fluid H in the centrifuge tube of magnetic bead, beat rear room temperature and leave standstill 5-10min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, suck liquid, retain magnetic bead, then toward containing adding 30-60 μ l working fluid I in the centrifuge tube of magnetic bead, centrifuge tube is placed on magnetic frame, carry out Beads enrichment, imbitition when magnetic bead is adsorbed onto centrifuge tube side, this liquid is gram-positive microorganism genomic dna, follow-up in 1% sepharose electrophoresis detection,
Described working fluid A is 10-50mg/ml N,O-Diacetylmuramidase;
Described working fluid B is mass concentration 10-20% sodium lauryl sulphate (SDS);
Described working fluid C is 20-50mg/ml Proteinase K;
Described working fluid D is 4-6mol/L sodium-chlor (Nacl);
Described working fluid E is volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described working fluid F is volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described working fluid G is volume ratio is Virahol: the suspension of magnetic bead suspension=24:1, described magnetic bead suspension is formed according to volume ratio 1:2 mixed configuration by magnetic bead and water, described magnetic bead is silica-based biomagnetic beads, particle diameter is 1um, buy from Wenzhou An Ke nanometer Science and Technology Ltd., production code member: AK1-G1000;
Described working fluid H is by final concentration 4.3-21.4mmol/L Tutofusin tris (Tris), final concentration 2.1-4.2mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), final concentration 42.9-85.7mmol/L Nacl and volume final concentration 60-80% dehydrated alcohol composition;
Described working fluid I is 5-10mmol/L Tutofusin tris (Tris), pH=7.0-9.0.
Final concentration of the present invention refers to the final concentration of solution in working fluid, such as described working fluid H is by final concentration 4.3-21.4mmol/L Tutofusin tris (Tris), final concentration 2.1-4.2mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), final concentration 42.9-85.7mmol/L NaCl and volume final concentration 60-80% dehydrated alcohol composition, refer to that the concentration of Tris in working fluid H is 4.3-21.4mmol/L, the concentration of EDTA in working fluid H is 2.1-4.2mmol/L, the concentration of NaCl in working fluid H is 42.9-85.7mmol/L, the volume final concentration of dehydrated alcohol in working fluid H is 60-80%.
Further, described working fluid A is 20mg/ml N,O-Diacetylmuramidase.
Further, described working fluid B is mass concentration 10% sodium lauryl sulphate.
Further, described working fluid C is 20mg/ml Proteinase K.
Further, described working fluid H is by final concentration 8.6mmol/L Tutofusin tris Tris, and final concentration 3.4mmol/L ethylenediamine tetraacetic acid (EDTA), final concentration 60mmol/L Nacl and volume final concentration 70% dehydrated alcohol form.
Further, described working fluid I is 8mmol/L Tutofusin tris, pH=8.0.
The present invention adopts above technical scheme, uses working fluid E and working fluid F to remove the impurity such as the albumen in cell solution, to obtain the higher gram-positive microorganism genomic dna of purity.Conventional gram-positive microorganism genome DNA extracting method mainly utilizes dehydrated alcohol low-temperature sludge genome, often need more than 10h consuming time, and the present invention is based on paramagnetic particle method extraction gram-positive microorganism genomic dna, silica-based magnetic bead surfaces is fixed with hydrophilic anions exchanger, the electronegative genomic DNA molecule of absorption that can be special, and other biological material is not adsorbed substantially, magnetic bead has magnetic, can by magnet adsorption, can the magnetic bead with genomic dna be adsorbed on centrifugal tube wall by magnetic frame, the nucleic acid in farthest recovery sample can be ensured, remove other impurity simultaneously, whole leaching process only needs can realize less than the time of 2h.
Compared with the gram-positive microorganism genome DNA extracting method of routine, the present invention not only saves a large amount of extraction time, simplifies experimental procedure simultaneously, can extract gram-positive microorganism genomic dna more effectively, fast, economically.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection figure of the gram-positive microorganism genomic dna that the inventive method is extracted: swimming lane 1,2, and 3 is the staphylococcus of extracting respectively, suis, actinomycetes genomic dna; Swimming lane 4 is the DL4500Marker of TAKARA company.
Embodiment
Based on a method for paramagnetic particle method rapid extraction gram-positive microorganism genomic dna, comprise the fragmentation of gram-positive bacteria cell, dissociating of DNA and extracting of foreign protein, paramagnetic particle method fast purifying genomic dna, described method specifically comprises the following steps:
1) fragmentation of gram-positive bacteria cell: get 3ml gram-positive microorganism incubated overnight liquid in centrifuge tube, supernatant is abandoned after the centrifugal 1-5min of each 10000-13000rpm/min, 0.2-0.5ml distilled water suspension precipitation is added in bacterial sediment, and add 50-80 μ l working fluid A, in 40 DEG C of insulation 10-50min, then add 30-60 μ l working fluid B and 5-10 μ l working fluid C, in 37 DEG C of insulation 30-60min after mixing, then add 0.4-0.6ml working fluid D, mix;
2) the extracting of free and foreign protein of DNA: in step 1) add 200-600 μ l working fluid E extracting in the mixed solution that obtains, then the centrifugal 10-30min of 10000-13000rpm/min, get a supernatant liquor in the 1.5ml centrifuge tube of sterilizing, add 200-600 μ l working fluid F extracting, then the centrifugal 10-30min of 10000-13000rpm/min, gets secondary supernatant liquor in aseptic 1.5ml centrifuge tube;
3) paramagnetic particle method fast purifying genomic dna: add with secondary supernatant liquor isopyknic working fluid G in the centrifuge tube containing secondary supernatant liquor, after concussion 10-30s, room temperature places 1-10min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, retain magnetic bead, then toward containing adding 0.5-1.5ml working fluid H in the centrifuge tube of magnetic bead, beat rear room temperature and leave standstill 5-10min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, suck liquid, retain magnetic bead, then toward containing adding 30-60 μ l working fluid I in the centrifuge tube of magnetic bead, centrifuge tube is placed on magnetic frame, carry out Beads enrichment, imbitition when magnetic bead is adsorbed onto centrifuge tube side, this liquid is gram-positive microorganism genomic dna, follow-up in 1% sepharose electrophoresis detection,
Described working fluid A is 10-50mg/ml N,O-Diacetylmuramidase;
Described working fluid B is mass concentration 10-20% sodium lauryl sulphate (SDS);
Described working fluid C is 20-50mg/ml Proteinase K;
Described working fluid D is 4-6mol/L sodium-chlor (Nacl);
Described working fluid E is volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described working fluid F is volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described working fluid G is volume ratio is Virahol: the suspension of magnetic bead suspension=24:1, and described magnetic bead suspension is formed according to volume ratio 1:2 mixed configuration by magnetic bead and water;
Described working fluid H is by final concentration 4.3-21.4mmol/L Tutofusin tris (Tris), final concentration 2.1-4.2mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), final concentration 42.9-85.7mmol/L Nacl and volume final concentration 60-80% dehydrated alcohol composition;
Described working fluid I is 5-10mmol/L Tutofusin tris (Tris), pH=7.0-9.0.
Embodiment 1
Based on a method for paramagnetic particle method rapid extraction gram-positive microorganism genomic dna, specifically comprise the following steps:
1) fragmentation of gram-positive bacteria cell: get 1ml staphylococcus incubated overnight liquid in 1.5ml centrifuge tube, supernatant is abandoned after the centrifugal 1min of 12000rpm/min, 1ml staphylococcus incubated overnight liquid is added again in centrifuge tube, supernatant is abandoned after the centrifugal 1-5min of 12000rpm/min, 1ml staphylococcus incubated overnight liquid is added again in centrifuge tube, supernatant is abandoned after the centrifugal 1min of 12000rpm/min, (be namely get 3ml staphylococcus incubated overnight liquid altogether, divide and be added to for 3 times in 1.5ml centrifuge tube, supernatant is abandoned) after the centrifugal 1min of each 12000rpm/min, then in bacterial sediment, add 0.25ml distilled water suspension precipitation, and add 75 μ l working fluid A, in 40 DEG C of insulation 30min, then 50 μ l working fluid B and 5 μ l working fluid C are added, in 37 DEG C of insulation 30min after mixing, then 0.45ml working fluid D is added, mix,
2) the extracting of free and foreign protein of DNA: in step 1) add 500 μ l working fluid E extractings in the mixed solution that obtains, then the centrifugal 10min of 12000rpm/min, get a supernatant liquor in the 1.5ml centrifuge tube of sterilizing, add 500 μ l working fluid F extractings, then the centrifugal 10min of 12000rpm/min, gets secondary supernatant liquor in aseptic 1.5ml centrifuge tube;
3) paramagnetic particle method fast purifying genomic dna: add with secondary supernatant liquor isopyknic working fluid G in the centrifuge tube containing secondary supernatant liquor, after concussion 30s, room temperature places 10min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, retain magnetic bead, then toward containing adding 1ml working fluid H in the centrifuge tube of magnetic bead, beat rear room temperature and leave standstill 5min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, suck liquid, retain magnetic bead, then toward containing adding 30 μ l working fluid I in the centrifuge tube of magnetic bead, centrifuge tube is placed on magnetic frame, carry out Beads enrichment, imbitition when magnetic bead is adsorbed onto centrifuge tube side, this liquid is aureus gene group DNA, follow-up in 1% sepharose electrophoresis detection,
Described working fluid A is 20mg/ml N,O-Diacetylmuramidase;
Described working fluid B is mass concentration 10% sodium lauryl sulphate (SDS);
Described working fluid C is 20mg/ml Proteinase K;
Described working fluid D is 5mol/L sodium-chlor (Nacl);
Described working fluid E is volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described working fluid F is volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described working fluid G is volume ratio is Virahol: the suspension of magnetic bead suspension=24:1, and described magnetic bead suspension is formed according to volume ratio 1:2 mixed configuration by magnetic bead and water;
Described working fluid H by final concentration 8.6mmol/L Tutofusin tris (Tris), final concentration 3.4mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), final concentration 60mmol/L Nacl and volume final concentration 70% dehydrated alcohol composition;
Described working fluid I is 8mmol/L Tutofusin tris (Tris), pH=8.0.
Adopt method same as described above and working fluid, extract suis genomic dna and actinomycetes genomic dna, follow-up in 1% sepharose electrophoresis detection.
Embodiment 2
Based on a method for paramagnetic particle method rapid extraction gram-positive microorganism genomic dna, specifically comprise the following steps:
1) fragmentation of gram-positive bacteria cell: get 1ml suis incubated overnight liquid in 1.5ml centrifuge tube, supernatant is abandoned after the centrifugal 5min of 10000rpm/min, 1ml suis incubated overnight liquid is added again in centrifuge tube, supernatant is abandoned after the centrifugal 5min of 10000rpm/min, 1ml suis incubated overnight liquid is added again in centrifuge tube, supernatant is abandoned after the centrifugal 5min of 10000rpm/min, (be namely get 3ml suis incubated overnight liquid altogether, divide and be added to for 3 times in 1.5ml centrifuge tube, supernatant is abandoned) after the centrifugal 5min of each 10000rpm/min, then in bacterial sediment, add 0.2ml distilled water suspension precipitation, and add 50 μ l working fluid A, in 40 DEG C of insulation 10min, then 30 μ l working fluid B and 10 μ l working fluid C are added, in 37 DEG C of insulation 60min after mixing, then 0.4ml working fluid D is added, mix,
2) the extracting of free and foreign protein of DNA: in step 1) add 200 μ l working fluid E extractings in the mixed solution that obtains, then the centrifugal 30min of 100000rpm/min, get a supernatant liquor in the 1.5ml centrifuge tube of sterilizing, add 200 μ l working fluid F extractings, then the centrifugal 30min of 10000rpm/min, gets secondary supernatant liquor in aseptic 1.5ml centrifuge tube;
3) paramagnetic particle method fast purifying genomic dna: add with secondary supernatant liquor isopyknic working fluid G in the centrifuge tube containing secondary supernatant liquor, after concussion 10s, room temperature places 1min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, retain magnetic bead, then toward containing adding 0.5ml working fluid H in the centrifuge tube of magnetic bead, beat rear room temperature and leave standstill 10min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, suck liquid, retain magnetic bead, then toward containing adding 45 μ l working fluid I in the centrifuge tube of magnetic bead, centrifuge tube is placed on magnetic frame, carry out Beads enrichment, imbitition when magnetic bead is adsorbed onto centrifuge tube side, this liquid is suis genomic dna, follow-up in 1% sepharose electrophoresis detection,
Described working fluid A is 10mg/ml N,O-Diacetylmuramidase;
Described working fluid B is mass concentration 15% sodium lauryl sulphate (SDS);
Described working fluid C is 35mg/ml Proteinase K;
Described working fluid D is 4mol/L sodium-chlor (Nacl);
Described working fluid E is volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described working fluid F is volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described working fluid G is volume ratio is Virahol: the suspension of magnetic bead suspension=24:1, and described magnetic bead suspension is formed according to volume ratio 1:2 mixed configuration by magnetic bead and water;
Described working fluid H by final concentration 4.3mmol/L Tutofusin tris (Tris), final concentration 2.1mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), final concentration 42.9mmol/L Nacl and volume final concentration 60% dehydrated alcohol composition;
Described working fluid I is 5mmol/L Tutofusin tris (Tris), pH=9.0.
Embodiment 3
Based on a method for paramagnetic particle method rapid extraction gram-positive microorganism genomic dna, specifically comprise the following steps:
1) fragmentation of gram-positive bacteria cell: get 1ml actinomycetes incubated overnight liquid in 1.5ml centrifuge tube, supernatant is abandoned after the centrifugal 2min of 13000rpm/min, 1ml actinomycetes incubated overnight liquid is added again in centrifuge tube, supernatant is abandoned after the centrifugal 2min of 13000rpm/min, 1ml actinomycetes incubated overnight liquid is added again in centrifuge tube, supernatant is abandoned after the centrifugal 2min of 13000rpm/min, (be namely get 3ml actinomycetes incubated overnight liquid altogether, divide and be added to for 3 times in 1.5ml centrifuge tube, supernatant is abandoned) after the centrifugal 2min of each 13000rpm/min, then in bacterial sediment, add 0.5ml distilled water suspension precipitation, and add 80 μ l working fluid A, in 40 DEG C of insulation 50min, then 60 μ l working fluid B and 10 μ l working fluid C are added, in 37 DEG C of insulation 45min after mixing, then 0.6ml working fluid D is added, mix,
2) the extracting of free and foreign protein of DNA: in step 1) add 600 μ l working fluid E extractings in the mixed solution that obtains, then the centrifugal 20min of 13000rpm/min, get a supernatant liquor in the 1.5ml centrifuge tube of sterilizing, add 600 μ l working fluid F extractings, then the centrifugal 20min of 13000rpm/min, gets secondary supernatant liquor in aseptic 1.5ml centrifuge tube;
3) paramagnetic particle method fast purifying genomic dna: add with secondary supernatant liquor isopyknic working fluid G in the centrifuge tube containing secondary supernatant liquor, after concussion 30s, room temperature places 10min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, retain magnetic bead, then toward containing adding 1.5ml working fluid H in the centrifuge tube of magnetic bead, beat rear room temperature and leave standstill 10min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, suck liquid, retain magnetic bead, then toward containing adding 60 μ l working fluid I in the centrifuge tube of magnetic bead, centrifuge tube is placed on magnetic frame, carry out Beads enrichment, imbitition when magnetic bead is adsorbed onto centrifuge tube side, this liquid is actinomycetes genomic dna, follow-up in 1% sepharose electrophoresis detection,
Described working fluid A is 50mg/ml N,O-Diacetylmuramidase;
Described working fluid B is mass concentration 20% sodium lauryl sulphate (SDS);
Described working fluid C is 50mg/ml Proteinase K;
Described working fluid D is 6mol/L sodium-chlor (Nacl);
Described working fluid E is volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described working fluid F is volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described working fluid G is volume ratio is Virahol: the suspension of magnetic bead suspension=24:1, and described magnetic bead suspension is formed according to volume ratio 1:2 mixed configuration by magnetic bead and water;
Described working fluid H by final concentration 21.4mmol/L Tutofusin tris (Tris), final concentration 4.2mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), final concentration 85.7mmol/L Nacl and volume final concentration 80% dehydrated alcohol composition;
Described working fluid I is 10mmol/L Tutofusin tris (Tris), pH=7.0.
Fig. 1 is the electrophoresis detection figure of the gram-positive microorganism genomic dna that the method for embodiment 1 is extracted: wherein, and swimming lane 1 is the test-results of the aureus gene group DNA extracted; Swimming lane 2 is the test-results of the suis genomic dna extracted; Swimming lane 3 is the test-results of the actinomycetes genomic dna extracted; Swimming lane 4 is the DL4500Marker of TAKARA company.The inventive method can extract Gram-positive genomic dna rapidly as can be seen from Figure 1.
The gram-positive microorganism genomic dna extracted, its purity OD
260nm/ OD
280nmratio is weighed, and purity is in table 1.
The purity of table 1 gram positive microbes DNA
As can be seen from Table 1, OD (260:280) scope of the gram-positive microorganism genomic dna utilizing present method to extract is between 1.78-1.81.Therefrom can find out, present method can extract the high gram-positive microorganism of purity as can be seen from Table 1.
Claims (6)
1. the method based on paramagnetic particle method rapid extraction gram-positive microorganism genomic dna, comprise the fragmentation of gram-positive bacteria cell, dissociating of DNA and extracting of foreign protein, paramagnetic particle method fast purifying genomic dna, is characterized in that: described method specifically comprises the following steps:
1) fragmentation of gram-positive bacteria cell: gram-positive microorganism incubated overnight liquid is added in centrifuge tube, supernatant is abandoned after the centrifugal 1-5min of 10000-13000rpm/min, 0.2-0.5ml distilled water and 50-80 μ l working fluid A is added in the bacterial sediment that every 3ml gram-positive microorganism incubated overnight liquid is corresponding, 10-50min is incubated in 40 ° of C, then 30-60 μ l working fluid B and 5-10 μ l working fluid C is added, 30-60min is incubated in 37 ° of C after mixing, then add 0.4-0.6ml working fluid D, mix;
2) dissociating of DNA and extracting of foreign protein: add 200-600 μ l working fluid E in the mixed solution that step 1) obtains, then the centrifugal 10-30min of 10000-13000rpm/min, get a supernatant liquor in the centrifuge tube of sterilizing, add 200-600 μ l working fluid F, then the centrifugal 10-30min of 10000-13000rpm/min, gets secondary supernatant liquor in aseptic centrifuge tube;
3) paramagnetic particle method fast purifying genomic dna: add with secondary supernatant liquor isopyknic working fluid G in the centrifuge tube containing secondary supernatant liquor, after concussion 10-30s, room temperature places 1-10min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, retain magnetic bead, then toward containing adding 0.5-1.5ml working fluid H in the centrifuge tube of magnetic bead, beat rear room temperature and leave standstill 5-10min, then centrifuge tube is placed on magnetic frame, carry out Beads enrichment, liquid is sucked when magnetic bead is adsorbed onto centrifuge tube side, suck liquid, retain magnetic bead, then toward containing adding 30-60 μ l working fluid I in the centrifuge tube of magnetic bead, centrifuge tube is placed on magnetic frame, carry out Beads enrichment, imbitition when magnetic bead is adsorbed onto centrifuge tube side, this liquid is gram-positive microorganism genomic dna,
Described working fluid A is 10-50 mg/ml N,O-Diacetylmuramidase;
Described working fluid B is mass concentration 10-20% sodium lauryl sulphate;
Described working fluid C is 20-50mg/ml Proteinase K;
Described working fluid D is 4-6mol/L sodium-chlor;
Described working fluid E is volume ratio is phenol: chloroform: the mixed solution of primary isoamyl alcohol=25:24:1;
Described working fluid F is volume ratio is chloroform: the mixed solution of primary isoamyl alcohol=24:1;
Described working fluid G is volume ratio is Virahol: the suspension of magnetic bead suspension=24:1, and described magnetic bead suspension is formed according to volume ratio 1:2 mixed configuration by magnetic bead and water;
Described working fluid H by final concentration 4.3-21.4 mmol/L Tutofusin tris, final concentration 2.1-4.2 mmol/L ethylenediamine tetraacetic acid (EDTA), final concentration 42.9-85.7 mmol/L Nacl and volume final concentration 60-80% dehydrated alcohol composition;
Described working fluid I is 5-10mmol/L Tutofusin tris, pH=7.0-9.0.
2. a kind of method based on paramagnetic particle method rapid extraction gram-positive microorganism genomic dna according to claim 1, is characterized in that: described working fluid A is 20 mg/ml N,O-Diacetylmuramidases.
3. a kind of method based on paramagnetic particle method rapid extraction gram-positive microorganism genomic dna according to claim 1, is characterized in that: described working fluid B is mass concentration 10% sodium lauryl sulphate.
4. a kind of method based on paramagnetic particle method rapid extraction gram-positive microorganism genomic dna according to claim 1, is characterized in that: described working fluid C is 20mg/ml Proteinase K.
5. a kind of method based on paramagnetic particle method rapid extraction gram-positive microorganism genomic dna according to claim 1, it is characterized in that: described working fluid H is by final concentration 8.6 mmol/L Tutofusin tris Tris, final concentration 3.4 mmol/L ethylenediamine tetraacetic acid (EDTA), final concentration 60mmol/L Nacl and volume final concentration 70% dehydrated alcohol composition.
6. a kind of method based on paramagnetic particle method rapid extraction gram-positive microorganism genomic dna according to claim 1, is characterized in that: described working fluid I is 8mmol/L Tutofusin tris, pH=8.0.
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| CN104975007A (en) * | 2015-07-28 | 2015-10-14 | 福建师范大学 | Method for extracting gram-negative bacteria genome with paramagnetic particle process |
| CN105018469A (en) * | 2015-07-28 | 2015-11-04 | 福建师范大学 | Kit for extracting silt microbe genome DNA (deoxyribonucleic acid) on basis of magnetic bead process |
| CN105154430A (en) * | 2015-07-28 | 2015-12-16 | 福建师范大学 | Kit for extracting gram-positive bacterium genome |
| CN106591284A (en) * | 2015-10-16 | 2017-04-26 | 中国检验检疫科学研究院 | Quick extraction kit for common pathogenic bacterium genomes in food and method |
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| CN105154430A (en) * | 2015-07-28 | 2015-12-16 | 福建师范大学 | Kit for extracting gram-positive bacterium genome |
| CN106591284A (en) * | 2015-10-16 | 2017-04-26 | 中国检验检疫科学研究院 | Quick extraction kit for common pathogenic bacterium genomes in food and method |
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