CN104212793B - 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, is related to the test kit that a kind of paramagnetic particle method extracts bacterial genomes DNA
And its extracting method.
Background technology
In Protocols in Molecular Biology, the association point of gene library, enzyme action, molecular cloning or full-length genome is either set up
Analysis, is required for extracting DNA profiling from sample.The integrity of the DNA concentration, purity and the primary structure that are extracted is directly affected
To follow-up study, therefore the quality of DNA is the primary factor of gene studiess success or failure.
In traditional DNA extraction method, centrifugal column method and phenol-chloroform method are topmost two methods, its DNA for extracting
Though quality is higher, detection time length, high labor intensive, it is most important that the toxic reagent such as chloroform used in experimentation
Very big injury is all caused to operator and ecological environment, and adopts magnetic bead for the isolation technics of carrier, without the need for centrifugation, without the need for connecing
Tactile toxic reagent, and it is simple to operation, it is easy to automatization is realized, the short time can be achieved with quick, the high-quality of sample DNA
Extraction, be important directions that following high flux extracts nucleic acid development, with incomparable excellent of traditional DNA extraction method
Gesture.
At present, external more well-known biotech company, such as Qiagen, Promega, Amresco is developed in succession
Go out paramagnetic particle method nucleic acid extraction kit or paramagnetic particle method high-throughput nucleic acid extraction apparatus, their product mostly operating procedure is simple,
The Nucleic acid quality of acquisition is high, complete fragment, but expensive；The well-known biotech company of domestic contrast, such as Tiangeng, Kang Weishi
The paramagnetic particle method nucleic acid extraction kit that discipline, Jin Maige etc. are developed, built-in magnetic bead is polydispersion magnetic bead, although magnetic response speed
Quickly, but magnetic bead sedimentation velocity to cause magnetic bead and buffer to act on also quickly, thus insufficient, this also becomes restriction its product and sends out
One principal element of exhibition.Therefore, built-in magnetic bead is developed at home for single dispersing magnetic bead and coordinates corresponding Laemmli buffer system Laemmli, make
The DNA fragmentation of acquisition is big, purity is high, steady quality reliability, and the paramagnetic particle method nucleic acid extraction product for meeting subsequent experimental requirement is compeled
In the eyebrows and eyelashes.
The content of the invention
The invention aims to overcome the deficiencies in the prior art, there is provided one kind extracts bacterial genomes using paramagnetic particle method
The test kit and its extracting method of DNA.
The purpose of the present invention is achieved through the following technical solutions：A kind of paramagnetic particle method extracts the examination of bacterial genomes DNA
Agent box, including buffer A, buffer B, buffer C, bead suspension D, buffer E, buffer F, seven kinds of components of buffer G；
The buffer A final concentration is consisted of：20-50mmol/L glucoses, 10-30mmol/L trishydroxymethylaminomethane
Hydrochlorate（Tris-HCl）, 5-20mmol/L ethylenediaminetetraacetic acid（EDTA）, solvent is autoclaving water (buffer A end PH=
8.0-8.5)；
The buffer B final concentration is consisted of：15-25mg/ml E.C. 3.4.21.64s, 1-5mol/L guanidine hydrochlorides, 5-25mmol/L lemons
Lemon acid sodium, 1-2% TritonX（Triton X-100）, 0.5-5mmol/L EDTA, solvent be autoclaving water (buffer B end
PH=4.0-5.5)；
The buffer C is into being grouped into：Ethanol solution；
The buffer E final concentrations are consisted of：1-5mol/L sodium acetates, volume fraction be 20-40% dehydrated alcohol, solvent
For autoclaving water (buffer E end PH=4.0-5.5)；
The buffer F final concentrations are consisted of：10-30mmol/L Tris-HCl、0.5-5mmol/L 0.5-5mmol/L
EDTA, 5-25mmol/L sodium citrate and dehydrated alcohol, the dehydrated alcohol of 0.3-0.7 L/L, solvent is that autoclaving water is (slow
Rush liquid F end PH=8.0-8.5)；
The buffer G final concentrations are consisted of：10-30mmol/L Tris-HCl, 0.5-5mmol/L EDTA, solvent is
Autoclaving water (buffer G end PH=8.0-8.5).
Bead suspension D is into being grouped into：Contain 50mg single dispersings in per 200mmol/L sodium-chloride water solutions
Fe3O4@SiO2- AEAPS nanometer magnetic beads.Single dispersing Fe3O4@SiO2- AEAPS nanometer magnetic beads are prepared by following steps：
（1）Solvent-thermal method prepares monodispersed ferriferrous oxide nano sphere；
（2）Using improved St ber methods, in ferriferrous oxide nano sphere outer cladding silicon dioxide, obtain monodispersed
Fe3O4@SiO2Nanosphere, specially：Weigh 0.1g steps（1）The ferriferrous oxide nano sphere for preparing, adds successively thereto
Enter 20ml deionized waters, 80ml dehydrated alcohol and ammonia that 1ml mass fractions are 28%, in the uniform backward mixed liquor of ultrasonic mixing
Add 1ml tetraethyl orthosilicates, room temperature（20°C）Lower mechanical agitation 6 hours；After completion of the reaction, resulting solution is centrifuged, and successively
Deionized water and ethanol purge 3 times, are obtained monodispersed coated with silica ferriferrous oxide nano sphere（Fe3O4@SiO2Receive
Rice ball）, by products therefrom in 60 °C of baking ovens dried for standby；
（3）Using silane coupler aminoethylaminopropyl polydimethylsiloxane to step（2）The Fe3O of acquisition4@SiO2Enter
Row silanization treatment, obtains Fe3O4@SiO2-AEAPS；Specially：Weigh 0.1g steps（2）The Fe3O4@SiO2 nanometers for obtaining
Ball, in being added to 20ml dimethylformamides, obtains solution A after ultrasonic disperse；By the aminoethylaminopropyl poly dimethyl of 10ml
After the dimethylformamide mix homogeneously of siloxanes and 20ml, add a certain amount of succinic anhydride that mixed solution pH value is maintained
3.9 ~ 4.1,60 °C of lower mechanical agitation 3 hours obtain solution B；20ml deionized waters will be added after solution A, B mixing, at 60 °
Continue mechanical agitation after 5 hours under C, resulting solution is centrifuged, and deionized water and ethanol purge 3 times successively, it is obtained single point
The coated with silica ferriferrous oxide nano sphere of scattered silanization（Fe3O4@SiO2-AEAPS）.
The method for extracting bacterial genomes DNA with test kit, comprises the steps：
（1）Take inoculum 1-1.5ml to be placed in 2.0ml EP pipes, 12000 rpm centrifugation 1-3min, collects thalline,
Add 150-300ul buffer As；
Note：If gram positive bacteria, 20 μ l lysozyme need to be continuously added after 150-300ul buffer As are added
（20mg/ml）Broken wall treatment is carried out, mixing is lashed or is vibrated and EP pipes are placed in into 37 °C of water-baths incubation 1h after mixing, period mixes number
It is secondary.
（2）To step（1）150-300ul buffer Bs, 45-65 °C of water-bath is added to be incubated 5-10min in EP pipes；
（3）To step（2）150-300ul buffer C are added in EP pipes after incubation, 5-10min is stood after mixing；
（4）To step（3）10-50ul bead suspensions D are added in EP pipes after standing, EP pipes are placed on magnetic frame,
Stand and remove liquid after 10-60s；
（5）By step（4）EP pipes are removed from magnetic frame, add 200-600ul buffer E, manage EP after mixing
It is placed on magnetic frame, stands and remove liquid after 10-60s, then EP pipes are removed from magnetic frame, adds 200-1000ul buffer
F, lashes EP pipes are placed on magnetic frame after mixing or vibration mixing, to stand and remove liquid after 10-60s, is stored at room temperature 5-
10min；
（6）By step（5）EP pipes are removed from magnetic frame, add 50-100ul buffer G, are placed in EP pipes after mixing
45-65 °C of water-bath is incubated 5-10min, EP pipes is placed on magnetic frame after taking-up supernatant is transferred to into collection after standing 10-60s
Guan Zhong, completes 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 bacterial genomes DNA
Test kit and its extracting method, be a kind of simple and effective method for extracting nucleic acid.Dispersed nano in test kit of the present invention
Magnetic bead, with more uniform size and shape, sedimentation velocity is slow in homemade buffer, is easy to magnetic bead fully to connect with nucleic acid
Touch, increase extraction efficiency, we are connected to the functional group that specifically can be had an effect with DNA in magnetic bead surfaces, with reversible
The characteristic of adsorption of DNA, then it is equipped with the buffer system of uniqueness so that test kit of the present invention is not needing any toxic solvent, is not required to
Be centrifuged repeatedly, the step such as vacuum filter or post separation, based on only combining magnetic bead by nucleic acid, just can reach and extract
Bacterial genomes DNA fragmentation it is big, purity is high, steady quality reliability, meet subsequent experimental requirement, greatly reduce experiment to work
Make the harm of personnel, and the particular/special requirement for reducing experimental facilitiess.
Description of the drawings
The agarose gel electrophoresis figure of the shigella dysenteriae genomic DNA that the test kit of Fig. 1 present invention is extracted；
The agarose gel electrophoresis figure of staphylococcus aureus gene group DNA that the test kit of Fig. 2 present invention is extracted.
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, the scope of the claims in the present invention are not constituted further
Restriction.
Embodiment 1, single dispersing Fe3O4@SiO2The preparation of-AEAPS nanometer magnetic beads.
（1）Monodispersed ferriferrous oxide nano sphere is prepared using solvent-thermal method, concrete preparation process is as follows：Weigh respectively
2.0g sodium acetates and 0.25g ferric chloride hexahydrates, in being added to 50ml ethylene glycol solutions, magnetic agitation is after 1 hour by body
System is transferred in reactor, is reacted 10 hours under 100 °C.After completion of the reaction, by gained dark solution be centrifuged, and spend successively from
Sub- water and ethanol purge 3 times, are obtained monodispersed ferriferrous oxide nano sphere（Fe3O4）, products therefrom is done in 60 °C of baking ovens
It is dry stand-by；
（2）Monodispersed coated with silica ferriferrous oxide nano sphere is prepared using improved St ber methods, concrete system
Standby process is as follows：Weigh 0.1g steps（1）The ferriferrous oxide nano sphere for preparing, sequentially adds thereto 20ml deionizations
Water, 80ml dehydrated alcohol and the ammonia that 1ml mass fractions are 28%, in the uniform backward mixed liquor of ultrasonic mixing the positive silicic acid of 1ml is added
Ethyl ester, mechanical agitation 6 hours under room temperature.After completion of the reaction, gained dark solution is centrifuged, and deionized water and ethanol successively
Cleaning 3 times, is obtained monodispersed coated with silica ferriferrous oxide nano sphere（Fe3O4@SiO2）, by products therefrom at 60 °C
Dried for standby in baking oven.
（3）Weigh 0.1g steps（2）The Fe for obtaining3O4@SiO2Nanosphere, in being added to 20ml dimethylformamides, ultrasound
Solution A is obtained after dispersion；The dimethylformamide mixing of the aminoethylaminopropyl polydimethylsiloxane of 10ml and 20ml is equal
After even, add a certain amount of succinic anhydride to cause mixed solution pH value to maintain 3.9 ~ 4.1,60 °C of lower mechanical agitation 3 hours, obtain
To solution B；20ml deionized waters will be added after solution A, B mixing, it is after continuing mechanical agitation 5 hours under 60 °C, gained is black
Color solution centrifugal, and deionized water and ethanol purge 3 times successively, are obtained the oxygen of coated with silica four of monodispersed silanization
Change three-iron nanosphere（Fe3O4@SiO2-AEAPS）, products therefrom is dried in 60 °C of baking ovens.
Embodiment 2：Application Example.
（1）Take fresh shigella dysenteriae culture fluid 1ml to be placed in 2.0ml EP pipes, 12000 rpm 1min centrifugations, collects thalline,
200ul buffer As are added, mixing is lashed, makes thalline resuspended；
（2）To step（1）200ul buffer Bs, 56 °C of water-baths is added to be incubated 10min in EP pipes；
（3）To step（2）200ul buffer C are added in EP pipes after incubation, mixing is lashed, 3min is stored at room temperature；
（4）To step（3）30ul bead suspensions D are added in EP pipes after standing, mixing is lashed, EP pipes are placed in into magnetic force
On frame, 30s is stood, liquid is carefully removed when magnetic bead adsorbs completely；
（5）By step（4）EP pipes are removed from magnetic frame, add 500ul buffer E, are lashed and are placed in EP pipes after mixing
On magnetic frame, 30s is stood, liquid is carefully removed when magnetic bead adsorbs completely, then EP pipes are removed from magnetic frame, added
1000ul buffer F, lash EP pipes are placed on magnetic frame after mixings, stand 30s, and liquid is carefully removed when magnetic bead adsorbs completely
Body, is stored at room temperature 10min；
（6）By step（5）EP pipes are removed from magnetic frame, add 50ul buffer G, are lashed and are placed in EP pipes after mixing
56 °C of water-baths, are incubated 5min, EP pipes are placed on magnetic frame after taking-up stand 30s, carefully that DNA is molten when magnetic bead adsorbs completely
Liquid is transferred in collecting pipe, is placed in -20 °C and is saved backup.
By the concentration and purity of genomic DNA Jing NanoDrop (ND-1000) the Detection and Extraction DNA for being extracted, wherein DNA
Purity is weighed with the ratio of OD260/OD280（It is generally acknowledged that the ratio of pure dna OD260/OD280 between 1.8-2.0 it
Between）, each sample does Parallel testing twice, and testing result is as shown in table 1.
Table 1
| DNA concentration (ng/ μ l) | OD260/OD280 | |
| Detection sample 1 | 559.5 | 1.83 |
| Detection sample 2 | 562.8 | 1.85 |
As shown in Table 1, this test kit is extracted bacterial genomes DNA extraction efficiency high, purity are high, steady quality can
Lean on, meet subsequent experimental requirement.The DNA for taking extraction does template, on the agarose gel of mass concentration 1% 200v voltages,
Under 100mA electric currents, electrophoresis 25min is taken a picture, it is seen that gene with ultraviolet gel imaging system (BIORAD-GelDoc 2000)
Group DNA fragmentation size is 204bp, and fragment integrity is good, as shown in Figure 1.Leftmost swimming lane is object of reference DL2000 in Fig. 1
(2000,1000,750,500,250,100)；Swimming lane 1 is negative control（Water）；Swimming lane 2 is positive control（cDNA）；Swimming lane 3,4
For the genomic DNA that test kit of the present invention is extracted.
Embodiment 3：Application Example.
（1）Take fresh staphylococcus aureuses culture fluid 1ml to be placed in 2.0ml EP pipes, 12000 rpm 1min centrifugations,
Collects thalline, adds 200ul buffer As, 20 μ l lysozyme（20mg/ml）, EP pipes are placed in into 37 °C of water-baths after vibration mixing and are incubated
1h is educated, period mixes 3 times；
（2）To step（1）200ul buffer Bs, 56 °C of water-baths is added to be incubated 10min in EP pipes；
（3）To step（2）200ul buffer C are added in EP pipes after incubation, mixing is lashed, 3min is stored at room temperature；
（4）To step（3）30ul bead suspensions D are added in EP pipes after standing, EP pipes are placed on magnetic frame, stood
30s, carefully removes liquid when magnetic bead adsorbs completely；
（5）By step（4）EP pipes are removed from magnetic frame, add 500ul buffer E, are lashed and are placed in EP pipes after mixing
On magnetic frame, 30s is stood, liquid is carefully removed when magnetic bead adsorbs completely, then EP pipes are removed from magnetic frame, added
1000ul buffer F, lash EP pipes are placed on magnetic frame after mixings, stand 30s, and liquid is carefully removed when magnetic bead adsorbs completely
Body, is stored at room temperature 10min；
（6）By step（5）EP pipes are removed from magnetic frame, add 50ul buffer G, are lashed and are placed in EP pipes after mixing
56 °C of water-baths, are incubated 5min, EP pipes are placed on magnetic frame after taking-up stand 30s, carefully that DNA is molten when magnetic bead adsorbs completely
Liquid is transferred in collecting pipe, is placed in -20 °C and is saved backup.
By the concentration and purity of genomic DNA Jing NanoDrop (ND-1000) the Detection and Extraction DNA for being extracted, wherein DNA
Purity is weighed with the ratio of OD260/OD280（It is generally acknowledged that the ratio of pure dna OD260/OD280 between 1.8-2.0 it
Between）, each sample does Parallel testing twice, and testing result is as shown in table 2.
Table 2
| DNA concentration (ng/ μ l) | OD260/OD280 | |
| Detection sample 1 | 338.8 | 1.96 |
| Detection sample 2 | 335.1 | 1.92 |
As shown in Table 2, this test kit is extracted bacterial genomes DNA extraction efficiency high, purity are high, steady quality can
Lean on, meet subsequent experimental requirement.The DNA for taking extraction does template, on the agarose gel of mass concentration 1% 200v voltages,
Under 100mA electric currents, electrophoresis 25min is taken a picture, it is seen that gene with ultraviolet gel imaging system (BIORAD-GelDoc 2000)
Group DNA fragmentation size is 279bp, and fragment integrity is good, as shown in Figure 2.Leftmost swimming lane is object of reference DM2000 in Fig. 2
(2000,1500,1000,750,500,250,100)；Swimming lane 1 is negative control（Water）；Swimming lane 2 is positive control（cDNA）；Swimming
Road 3,4 is the genomic DNA that test kit of the present invention is extracted.
Claims (2)
1. a kind of paramagnetic particle method extracts the test kit of bacterial genomes DNA, it is characterised in that：It includes buffer A, buffer B, delays
Rush liquid C, bead suspension D, buffer E, buffer F, seven kinds of components of buffer G；
The buffer A final concentration is consisted of：20-50mmol/L glucoses, 10-30mmol/L trishydroxymethylaminomethane hydrochloric acid
Salt, 5-20mmol/L ethylenediaminetetraacetic acid, solvent be autoclaving water, buffer A end PH=8.0-8.5；
The buffer B final concentration is consisted of：15-25mg/ml E.C. 3.4.21.64s, 1-5mol/L guanidine hydrochlorides, 5-25mmol/L citric acids
Sodium, 1-2% TritonX, 0.5-5mmol/L EDTA, solvent be autoclaving water, buffer B end PH=4.0-5.5；
The buffer C is into being grouped into：Ethanol solution；
The buffer E final concentrations are consisted of：1-5mol/L sodium acetates, volume fraction is 20-40% dehydrated alcohol, and solvent is height
Pressure aquesterilisa, buffer E end PH=4.0-5.5；
The buffer F final concentrations are consisted of：10-30mmol/L Tris-HCl、0.5-5mmol/L EDTA、5-25mmol/L
The dehydrated alcohol of sodium citrate and 0.3-0.7 L/L, solvent be autoclaving water, buffer F end PH=8.0-8.5；
The buffer G final concentrations are consisted of：10-30mmol/L Tris-HCl, 0.5-5mmol/L EDTA, solvent is high pressure
Aquesterilisa, buffer G end PH=8.0-8.5；
Bead suspension D is into being grouped into：Contain 50mg single dispersing Fe in per 200mmol/L sodium-chloride water solutions3O4@
SiO2- AEAPS nanometer magnetic beads；
The single dispersing Fe3O4@SiO2- AEAPS nanometer magnetic beads are prepared by following steps：
（1）Solvent-thermal method prepares monodispersed ferriferrous oxide nano sphere；
（2）Using improved St ber methods, in ferriferrous oxide nano sphere outer cladding silicon dioxide, monodispersed Fe is obtained3O4@
SiO2Nanosphere, specially：Weigh 0.1g steps（1）The ferriferrous oxide nano sphere for preparing, sequentially adds thereto
20ml deionized waters, 80ml dehydrated alcohol and the ammonia that 1ml mass fractions are 28%, add in the uniform backward mixed liquor of ultrasonic mixing
Enter 1ml tetraethyl orthosilicates, 20 °C of lower mechanical agitation 6 hours；After completion of the reaction, resulting solution is centrifuged, and uses deionization successively
Water and ethanol purge 3 times, are obtained monodispersed coated with silica ferriferrous oxide nano sphere, by products therefrom in 60 °C of baking ovens
Middle dried for standby；
（3）Using silane coupler aminoethylaminopropyl polydimethylsiloxane to step（2）The Fe3O of acquisition4@SiO2Carry out silicon
Alkanisation process, obtains Fe3O4@SiO2-AEAPS；Specially：Weigh 0.1g steps（2）The Fe for obtaining3O4@SiO2Nanosphere, adds
To in 20ml dimethylformamides, solution A is obtained after ultrasonic disperse；By the aminoethylaminopropyl polydimethylsiloxane of 10ml and
After the dimethylformamide mix homogeneously of 20ml, add a certain amount of succinic anhydride cause mixed solution pH value maintain 3.9 ~
4.1,60 °C of lower mechanical agitation 3 hours, obtain solution B；Will solution A, B mixing after add 20ml deionized waters, under 60 °C after
After continuous mechanical agitation 5 hours, resulting solution is centrifuged, and deionized water and ethanol purge 3 times successively, monodispersed silicon is obtained
The coated with silica ferriferrous oxide nano sphere Fe of alkanisation3O4@SiO2-AEAPS。
2. the method that the test kit described in a kind of claim 1 extracts bacterial genomes DNA, it is characterised in that including following step
Suddenly：
（1）Take tested bacteria culture fluid 1-1.5ml to be placed in 2.0ml EP pipes, 12000 rpm centrifugation 1-3min, collects thalline,
Add 150-300 μ l buffer As；
（2）To step（1）150-300 μ l buffer Bs, 45-65 °C of water-bath is added to be incubated 5-10min in EP pipes；
（3）To step（2）150-300 μ l buffer C are added in EP pipes after incubation, 5-10min is stood after mixing；
（4）To step（3）10-50 μ l bead suspensions D are added in EP pipes after standing, EP pipes are placed on magnetic frame, stood
Liquid is removed after 10-60s；
（5）By step（4）EP pipes are removed from magnetic frame, add 200-600 μ l buffer E, are placed in EP pipes after mixing
On magnetic frame, to stand and remove liquid after 10-60s, then EP pipes are removed from magnetic frame, add 200-1000 μ l buffer F, take out
Beat to mix or vibrate EP pipes are placed on magnetic frame after mixing, to stand and remove liquid after 10-60s, be stored at room temperature 5-10min；
（6）By step（5）EP pipes are removed from magnetic frame, add 50-100 μ l buffer G, and EP pipes are placed in into 45-65 ° after mixing
C water-baths are incubated 5-10min, EP pipes are placed on magnetic frame after taking-up supernatant is transferred in collecting pipe after standing 10-60s,
Complete the extraction of DNA of bacteria.
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