CN104212793A - Kit for magnetic bead method for bacterial genome DNA extraction and extraction method thereof - Google Patents
Kit for magnetic bead method for bacterial genome DNA extraction and extraction method thereof Download PDFInfo
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Abstract
The invention discloses a kit for a magnetic bead method for bacterial genome DNA extraction and an extraction method thereof. The kit includes the following seven components: a buffer solution A, a buffer solution B, a buffer solution C, a magnetic bead suspension solution D, a buffer solution E, a buffer solution F and a buffer solution G. composition of the magnetic bead suspension solution D is 50mg of monodisperse Fe3O4 @ SiO2 AEAPS nanometer magnetic beads in each 200 mmol / L sodium chloride aqueous solution. The extraction method for the kit comprises six steps: thallus re-suspension, cracking, nucleic acid deposition, magnetic bead adsorption, washing and elution. The kit provided by the invention adopts efficient monodisperse nano magnetic beads combined with a unique buffer system, so that the extracted bacterial genome DNA has large fragment, high purity and stable and reliable quality, and can meet the requirements of the follow-up experiments.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to test kit and extracting method thereof that a kind of paramagnetic particle method extracts bacterial genomes DNA.
Background technology
In Protocols in Molecular Biology, no matter set up gene library, enzyme is cut, molecular cloning or whole-genome association, all need to extract DNA profiling from sample.The integrity of DNA concentration, purity and the primary structure extracted directly has influence on follow-up study, and therefore the quality of DNA is the primary factor of gene studies success or failure.
In traditional DNA extraction method, centrifugal column method and phenol-chloroform method are topmost two kinds of methods, though its DNA quality extracted is higher, but detection time is long, labour intensity is large, the most important thing is that the toxic reagents such as the chloroform used in experimentation all cause very large injury to operator and ecotope, and adopt magnetic bead to be the isolation technique of carrier, without the need to centrifugal, without the need to contacting toxic reagent, and it is simple to operation, be easy to realize automatization, short period of time just can realize the quick of sample DNA, high-quality extraction, it is the important directions that following high-throughput extracts nucleic acid development, there is the advantage that traditional DNA extraction method is incomparable.
At present, external more well-known biotech company, as Qiagen, Promega, Amresco etc. have developed paramagnetic particle method nucleic acid extraction kit or paramagnetic particle method high-throughput nucleic acid extraction apparatus in succession, mostly operation steps Nucleic acid quality that is simple, that obtain is high for their product, complete fragment, but expensive; The biotech company that domestic contrast is well-known, if sky root, health are the paramagnetic particle method nucleic acid extraction kit that century, Jin Maige etc. develop, built-in magnetic bead is polydispersion magnetic bead, although magnetic response speed is very fast, but magnetic bead settling velocity is also very fast, cause thus magnetic bead and damping fluid effect insufficient, this also become restriction its production development a principal element.Therefore, develop built-in magnetic bead at home and be single dispersing magnetic bead and coordinate corresponding Laemmli buffer system Laemmli, make that the DNA fragmentation of acquisition is large, purity is high, steady quality is reliable, meet the paramagnetic particle method nucleic acid extraction product that subsequent experimental requires extremely urgent.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of test kit and the extracting method thereof that adopt paramagnetic particle method extraction bacterial genomes DNA are provided.
The object of the invention is to be achieved through the following technical solutions: a kind of paramagnetic particle method extracts the test kit of bacterial genomes DNA, comprises buffer A, buffer B, damping fluid C, bead suspension D, damping fluid E, damping fluid F, damping fluid G seven kinds of components;
Described buffer A final concentration consists of: 20-50mmol/L glucose, 10-30mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl), 5-20mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), and solvent is autoclaving water (the whole PH=8.0-8.5 of buffer A);
Described buffer B final concentration consists of: 15-25mg/ml Proteinase K, 1-5mol/L Guanidinium hydrochloride, 5-25mmol/L Trisodium Citrate, 1-2% Triton (Triton X-100), 0.5-5mmol/L EDTA, and solvent is autoclaving water (the whole PH=4.0-5.5 of buffer B);
Described damping fluid C composition consists of: ethanol solution;
Described damping fluid E final concentration consists of: 1-5mol/L sodium-acetate, and volume fraction is 20-40% dehydrated alcohol, and solvent is autoclaving water (the whole PH=4.0-5.5 of damping fluid E);
Described damping fluid F final concentration consists of: 10-30mmol/L Tris-HCl, 0.5-5mmol/L 0.5-5mmol/L EDTA, 5-25mmol/L Trisodium Citrate and dehydrated alcohol, the dehydrated alcohol of 0.3-0.7 L/L, solvent is autoclaving water (the whole PH=8.0-8.5 of damping fluid F);
Described damping fluid G final concentration consists of: 10-30mmol/L Tris-HCl, 0.5-5mmol/L EDTA, and solvent is autoclaving water (the whole PH=8.0-8.5 of damping fluid G).
Described bead suspension D composition consists of: containing 50mg single dispersing Fe in every 200mmol/L sodium chloride aqueous solution
3o
4@SiO
2-AEAPS nanometer magnetic bead.Single dispersing Fe
3o
4@SiO
2-AEAPS nanometer magnetic bead is prepared by following steps:
(1) solvent-thermal method prepares monodispersed ferriferrous oxide nano sphere;
(2) adopt the St ber method improved, at ferriferrous oxide nano sphere outer cladding silicon-dioxide, obtain monodispersed Fe
3o
4@SiO
2nanometer ball, be specially: take the ferriferrous oxide nano sphere that 0.1g step (1) prepares, adding 20ml deionized water, 80ml dehydrated alcohol and 1ml massfraction wherein is successively the ammoniacal liquor of 28%, ultrasonic mixing in backward mixed solution adds 1ml tetraethoxy, mechanical stirring 6 hours under room temperature (20 ° of C); After completion of the reaction, by gained solution centrifugal, and use deionized water and ethanol purge 3 times successively, obtained monodispersed coated with silica ferriferrous oxide nano sphere (Fe
3o
4@SiO
2nanometer ball), by products therefrom dried for standby in 60 ° of C baking ovens;
(3) Fe3O that silane coupling agent aminoethylaminopropyl polydimethylsiloxane obtains step (2) is used
4@SiO
2carry out silanization treatment, obtain Fe
3o
4@SiO
2-AEAPS; Be specially: take the Fe3O4@SiO2 nanometer ball that 0.1g step (2) obtains, join in 20ml dimethyl formamide, after ultrasonic disperse, obtain solution A; After the aminoethylaminopropyl polydimethylsiloxane of 10ml and the dimethyl formamide of 20ml being mixed, add a certain amount of Succinic anhydried and make mixing solutions pH value maintain 3.9 ~ 4.1, under 60 ° of C, mechanical stirring 3 hours, obtains solution B; 20ml deionized water is added by after solution A, B mixing, mechanical stirring is continued after 5 hours under 60 ° of C, by gained solution centrifugal, and use deionized water and ethanol purge 3 times successively, the coated with silica ferriferrous oxide nano sphere (Fe3O4 SiO2-AEAPS) of obtained monodispersed silanization.
Extract the method for bacterial genomes DNA with test kit, comprise the steps:
(1) get inoculum 1-1.5ml and be placed in 2.0ml EP pipe, the centrifugal 1-3min of 12000 rpm, collect thalline, add 150-300ul buffer A;
Attention: if gram-positive microorganism, need continue to add 20 μ l N,O-Diacetylmuramidases (20mg/ml) and carry out broken wall treatment after adding 150-300ul buffer A, and after lashing mixing or vibration mixing, EP pipe is placed in 37 ° of C water-baths and hatches 1h, period mixing for several times.
(2) in step (1) EP pipe, add 150-300ul buffer B, 5-10min is hatched in 45-65 ° of C water-bath;
(3) add 150-300ul damping fluid C in the EP pipe after hatching to step (2), after mixing, leave standstill 5-10min;
(4) add 10-50ul bead suspension D in the EP pipe after leaving standstill to step (3), EP pipe is placed on magnetic frame, after leaving standstill 10-60s, remove liquid;
(5) step (4) EP pipe is taken off from magnetic frame, add 200-600ul damping fluid E, after mixing mixing, EP pipe is placed on magnetic frame, liquid is removed after leaving standstill 10-60s, again EP pipe is taken off from magnetic frame, add 200-1000ul damping fluid F, after lashing mixing or vibration mixing, EP pipe is placed on magnetic frame, remove liquid after leaving standstill 10-60s, room temperature leaves standstill 5-10min;
(6) step (5) EP pipe is taken off from magnetic frame, add 50-100ul damping fluid G, after mixing, EP pipe is placed in 45-65 ° of C water-bath and hatches 5-10min, after taking-up, EP pipe is placed in after magnetic frame leaves standstill 10-60s and supernatant liquor is transferred to collection tube, complete the extraction of DNA of bacteria.
Compared with prior art, the invention has the beneficial effects as follows: paramagnetic particle method disclosed by the invention extracts test kit and the extracting method thereof of bacterial genomes DNA, is a kind of simple and effective method for extracting nucleic acid.Dispersed nano magnetic bead in test kit of the present invention, there is more homogeneous size and shape, in homemade damping fluid, settling velocity is slow, be convenient to magnetic bead fully contact with nucleic acid, increase extraction efficiency, we are connected to the functional group that can have an effect with DNA specifically in magnetic bead surfaces, there is the characteristic of reversible adsorption DNA, be equipped with unique buffering system again, make test kit of the present invention without any need for noxious solvent, do not need repeatedly centrifugal, the steps such as vacuum filtration or post separation, only in conjunction with based on magnetic bead by nucleic acid, just the bacterial genomes DNA fragmentation extracted can be reached large, purity is high, steady quality is reliable, meet subsequent experimental requirement, greatly reduce the harm of experiment to staff, and reduce the particular requirement of experimental installation.
Accompanying drawing explanation
The agarose gel electrophoresis figure of the shigella genomic dna that Fig. 1 test kit of the present invention extracts;
The agarose gel electrophoresis figure of the staphylococcus aureus gene group DNA that Fig. 2 test kit of the present invention extracts.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, the scope of the claims in the present invention are not formed and further limit.
embodiment 1, single dispersing Fe
3
o
4
@SiO
2
the preparation of-AEAPS nanometer magnetic bead.
(1) solvent-thermal method is adopted to prepare monodispersed ferriferrous oxide nano sphere, concrete preparation process is as follows: take 2.0g sodium-acetate and 0.25g ferric chloride hexahydrate respectively, join in 50ml ethylene glycol solution, system was transferred in reactor after 1 hour by magnetic agitation, reacted 10 hours under 100 ° of C.After completion of the reaction, by centrifugal for gained dark solution, and use deionized water and ethanol purge 3 times successively, obtained monodispersed ferriferrous oxide nano sphere (Fe
3o
4), by products therefrom dried for standby in 60 ° of C baking ovens;
(2) adopt the St ber legal system improved for monodispersed coated with silica ferriferrous oxide nano sphere, concrete preparation process is as follows: take the ferriferrous oxide nano sphere that 0.1g step (1) prepares, adding 20ml deionized water, 80ml dehydrated alcohol and 1ml massfraction wherein is successively the ammoniacal liquor of 28%, ultrasonic mixing in backward mixed solution adds 1ml tetraethoxy, mechanical stirring 6 hours under room temperature.After completion of the reaction, by centrifugal for gained dark solution, and use deionized water and ethanol purge 3 times successively, obtained monodispersed coated with silica ferriferrous oxide nano sphere (Fe
3o
4@SiO
2), by products therefrom dried for standby in 60 ° of C baking ovens.
(3) Fe that 0.1g step (2) obtains is taken
3o
4@SiO
2nanometer ball, joins in 20ml dimethyl formamide, obtains solution A after ultrasonic disperse; After the aminoethylaminopropyl polydimethylsiloxane of 10ml and the dimethyl formamide of 20ml being mixed, add a certain amount of Succinic anhydried and make mixing solutions pH value maintain 3.9 ~ 4.1, under 60 ° of C, mechanical stirring 3 hours, obtains solution B; 20ml deionized water is added by after solution A, B mixing, mechanical stirring is continued after 5 hours under 60 ° of C, by centrifugal for gained dark solution, and use deionized water and ethanol purge 3 times successively, the coated with silica ferriferrous oxide nano sphere (Fe of obtained monodispersed silanization
3o
4@SiO
2-AEAPS), products therefrom is dry in 60 ° of C baking ovens.
embodiment 2: Application Example.
(1) get fresh shigella nutrient solution 1ml and be placed in 2.0ml EP pipe, 12000 rpm 1min are centrifugal, collect thalline, add 200ul buffer A, lash mixing, make thalline resuspended;
(2) in step (1) EP pipe, add 200ul buffer B, 56 ° of C water-baths, hatch 10min;
(3) add 200ul damping fluid C in the EP pipe after hatching to step (2), lash mixing, room temperature leaves standstill 3min;
(4) add 30ul bead suspension D in the EP pipe after leaving standstill to step (3), lash mixing, EP pipe is placed on magnetic frame, leave standstill 30s, when magnetic bead adsorbs completely, carefully remove liquid;
(5) step (4) EP pipe is taken off from magnetic frame, add 500ul damping fluid E, after lashing mixing, EP pipe is placed on magnetic frame, leaves standstill 30s, when magnetic bead adsorbs completely, carefully remove liquid, again EP pipe is taken off from magnetic frame, add 1000ul damping fluid F, after lashing mixing, EP pipe is placed on magnetic frame, leave standstill 30s, when magnetic bead adsorbs completely, carefully remove liquid, room temperature leaves standstill 10min;
(6) step (5) EP pipe is taken off from magnetic frame, add 50ul damping fluid G, after lashing mixing, EP pipe is placed in 56 ° of C water-baths, hatch 5min, after taking-up, EP pipe is placed on magnetic frame and leaves standstill 30s, carefully DNA solution is transferred in collection tube when magnetic bead adsorbs completely, is placed in-20 ° of C and saves backup.
By extracted genomic dna through the concentration of NanoDrop (ND-1000) Detection and Extraction DNA and purity, wherein the ratio of DNA purity OD260/OD280 is weighed (it is generally acknowledged that the ratio of pure dna OD260/OD280 is between 1.8-2.0), each sample does twice Parallel testing, and detected result is as shown in table 1.
Table 1
| ? | DNA concentration (ng/ μ l) | OD260/OD280 |
| Detect sample 1 | 559.5 | 1.83 |
| Detect sample 2 | 562.8 | 1.85 |
As shown in Table 1, the bacterial genomes DNA extraction efficiency that this test kit extracts is high, purity is high, steady quality is reliable, meets subsequent experimental requirement.The DNA getting extraction does template, on the sepharose of mass concentration 1% under 200v voltage, 100mA electric current, electrophoresis 25min, take a picture with ultraviolet gel imaging system (BIORAD-GelDoc 2000), visible genetic group DNA fragmentation size is 204bp, fragment integrity is good, as shown in Figure 1.In Fig. 1, leftmost swimming lane is object of reference DL2000 (2000,1000,750,500,250,100); Swimming lane 1 is negative control (water); Swimming lane 2 is positive control (cDNA); The genomic dna that swimming lane 3,4 extracts for test kit of the present invention.
embodiment 3: Application Example.
(1) get fresh streptococcus aureus nutrient solution 1ml and be placed in 2.0ml EP pipe, 12000 rpm 1min are centrifugal, collect thalline, add 200ul buffer A, 20 μ l N,O-Diacetylmuramidases (20mg/ml), after vibration mixing, EP pipe is placed in 37 ° of C water-baths and hatches 1h, period mixes 3 times;
(2) in step (1) EP pipe, add 200ul buffer B, 56 ° of C water-baths, hatch 10min;
(3) add 200ul damping fluid C in the EP pipe after hatching to step (2), lash mixing, room temperature leaves standstill 3min;
(4) add 30ul bead suspension D in the EP pipe after leaving standstill to step (3), EP pipe is placed on magnetic frame, leave standstill 30s, when magnetic bead adsorbs completely, carefully remove liquid;
(5) step (4) EP pipe is taken off from magnetic frame, add 500ul damping fluid E, after lashing mixing, EP pipe is placed on magnetic frame, leaves standstill 30s, when magnetic bead adsorbs completely, carefully remove liquid, again EP pipe is taken off from magnetic frame, add 1000ul damping fluid F, after lashing mixing, EP pipe is placed on magnetic frame, leave standstill 30s, when magnetic bead adsorbs completely, carefully remove liquid, room temperature leaves standstill 10min;
(6) step (5) EP pipe is taken off from magnetic frame, add 50ul damping fluid G, after lashing mixing, EP pipe is placed in 56 ° of C water-baths, hatch 5min, after taking-up, EP pipe is placed on magnetic frame and leaves standstill 30s, carefully DNA solution is transferred in collection tube when magnetic bead adsorbs completely, is placed in-20 ° of C and saves backup.
By extracted genomic dna through the concentration of NanoDrop (ND-1000) Detection and Extraction DNA and purity, wherein the ratio of DNA purity OD260/OD280 is weighed (it is generally acknowledged that the ratio of pure dna OD260/OD280 is between 1.8-2.0), each sample does twice Parallel testing, and detected result is as shown in table 2.
Table 2
| ? | DNA concentration (ng/ μ l) | OD260/OD280 |
| Detect sample 1 | 338.8 | 1.96 |
| Detect sample 2 | 335.1 | 1.92 |
As shown in Table 2, the bacterial genomes DNA extraction efficiency that this test kit extracts is high, purity is high, steady quality is reliable, meets subsequent experimental requirement.The DNA getting extraction does template, on the sepharose of mass concentration 1% under 200v voltage, 100mA electric current, electrophoresis 25min, take a picture with ultraviolet gel imaging system (BIORAD-GelDoc 2000), visible genetic group DNA fragmentation size is 279bp, fragment integrity is good, as shown in Figure 2.In Fig. 2, leftmost swimming lane is object of reference DM2000 (2000,1500,1000,750,500,250,100); Swimming lane 1 is negative control (water); Swimming lane 2 is positive control (cDNA); The genomic dna that swimming lane 3,4 extracts for test kit of the present invention.
Claims (3)
1. paramagnetic particle method extracts a test kit of bacterial genomes DNA, it is characterized in that: it comprises buffer A, buffer B, damping fluid C, bead suspension D, damping fluid E, damping fluid F, damping fluid G seven kinds of components;
Described buffer A final concentration consists of: 20-50mmol/L glucose, 10-30mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl), 5-20mmol/L ethylenediamine tetraacetic acid (EDTA) (EDTA), and solvent is autoclaving water (the whole PH=8.0-8.5 of buffer A);
Described buffer B final concentration consists of: 15-25mg/ml Proteinase K, 1-5mol/L Guanidinium hydrochloride, 5-25mmol/L Trisodium Citrate, 1-2% Triton (Triton X-100), 0.5-5mmol/L EDTA, and solvent is autoclaving water (the whole PH=4.0-5.5 of buffer B);
Described damping fluid C composition consists of: ethanol solution;
Described damping fluid E final concentration consists of: 1-5mol/L sodium-acetate, and volume fraction is 20-40% dehydrated alcohol, and solvent is autoclaving water (the whole PH=4.0-5.5 of damping fluid E);
Described damping fluid F final concentration consists of: 10-30mmol/L Tris-HCl, 0.5-5mmol/L 0.5-5mmol/L EDTA, 5-25mmol/L Trisodium Citrate and dehydrated alcohol, the dehydrated alcohol of 0.3-0.7 L/L, solvent is autoclaving water (the whole PH=8.0-8.5 of damping fluid F);
Described damping fluid G final concentration consists of: 10-30mmol/L Tris-HCl, 0.5-5mmol/L EDTA, and solvent is autoclaving water (the whole PH=8.0-8.5 of damping fluid G);
Described bead suspension D composition consists of: containing 50mg single dispersing Fe in every 200mmol/L sodium chloride aqueous solution
3o
4@SiO
2-AEAPS nanometer magnetic bead.
2. paramagnetic particle method according to claim 1 extracts the test kit of bacterial genomes DNA, it is characterized in that, described single dispersing Fe
3o
4@SiO
2-AEAPS nanometer magnetic bead is prepared by following steps:
(1) solvent-thermal method prepares monodispersed ferriferrous oxide nano sphere;
(2) adopt the St ber method improved, at ferriferrous oxide nano sphere outer cladding silicon-dioxide, obtain monodispersed Fe
3o
4@SiO
2nanometer ball, be specially: take the ferriferrous oxide nano sphere that 0.1g step (1) prepares, adding 20ml deionized water, 80ml dehydrated alcohol and 1ml massfraction wherein is successively the ammoniacal liquor of 28%, ultrasonic mixing in backward mixed solution adds 1ml tetraethoxy, mechanical stirring 6 hours under room temperature (20 ° of C); After completion of the reaction, by gained solution centrifugal, and use deionized water and ethanol purge 3 times successively, obtained monodispersed coated with silica ferriferrous oxide nano sphere (Fe
3o
4@SiO
2nanometer ball), by products therefrom dried for standby in 60 ° of C baking ovens;
(3) Fe3O that silane coupling agent aminoethylaminopropyl polydimethylsiloxane obtains step (2) is used
4@SiO
2carry out silanization treatment, obtain Fe
3o
4@SiO
2-AEAPS; Be specially: take the Fe that 0.1g step (2) obtains
3o
4@SiO
2nanometer ball, joins in 20ml dimethyl formamide, obtains solution A after ultrasonic disperse; After the aminoethylaminopropyl polydimethylsiloxane of 10ml and the dimethyl formamide of 20ml being mixed, add a certain amount of Succinic anhydried and make mixing solutions pH value maintain 3.9 ~ 4.1, under 60 ° of C, mechanical stirring 3 hours, obtains solution B; Add 20ml deionized water by after solution A, B mixing, under 60 ° of C, continue mechanical stirring after 5 hours, by gained solution centrifugal, and use deionized water and ethanol purge 3 times successively, the coated with silica ferriferrous oxide nano sphere (Fe of obtained monodispersed silanization
3o
4@SiO
2-AEAPS).
3. test kit according to claim 1 extracts a method of bacterial genomes DNA, it is characterized in that, comprises the steps:
(1) get tested bacteria nutrient solution 1-1.5ml and be placed in 2.0ml EP pipe, the centrifugal 1-3min of 12000 rpm, collect thalline, add 150-300ul buffer A;
(2) in step (1) EP pipe, add 150-300ul buffer B, 5-10min is hatched in 45-65 ° of C water-bath;
(3) add 150-300ul damping fluid C in the EP pipe after hatching to step (2), after mixing, leave standstill 5-10min;
(4) add 10-50ul bead suspension D in the EP pipe after leaving standstill to step (3), EP pipe is placed on magnetic frame, after leaving standstill 10-60s, remove liquid;
(5) step (4) EP pipe is taken off from magnetic frame, add 200-600ul damping fluid E, after mixing mixing, EP pipe is placed on magnetic frame, liquid is removed after leaving standstill 10-60s, again EP pipe is taken off from magnetic frame, add 200-1000ul damping fluid F, after lashing mixing or vibration mixing, EP pipe is placed on magnetic frame, remove liquid after leaving standstill 10-60s, room temperature leaves standstill 5-10min;
(6) step (5) EP pipe is taken off from magnetic frame, add 50-100ul damping fluid G, after mixing, EP pipe is placed in 45-65 ° of C water-bath and hatches 5-10min, after taking-up, EP pipe is placed in after magnetic frame leaves standstill 10-60s and supernatant liquor is transferred to collection tube, complete the extraction of DNA of bacteria.
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