CN1299413A - Glass microfiber column and method for the prepn. and purification of plasmid DNA using the same - Google Patents

Abstract

Disclosed are glass microfiber column kits for plasmid DNA preparation and DNA purification and the methods for preparing and purifying DNA. The column kits use resins which are formed of borosilicate glass microfiber membranes and/or particles. They are useful to prepare a large quantity of highly pure plasmid DNAs from bacteria cultures and to purify DNA from agarose gels and polyacryl amide gels, simply and quickly. The DNA preparations are so highly pure that they can be applied for various higher experiments, including gene cloning, transfection into mammalian cells and transduction, base sequencing analysis, PCR, radio-labeled DNA probe quantification, cell proliferation bioassay using radio-labeled DNA, etc.

Description

Glass microfiber column and with its preparation and the method for plasmid DNA purification

Technical field

The present invention relates to glass microfiber (GF) post and with its preparation and the method for plasmid DNA purification.More particularly, the present invention relates to borosilicate or silicate glass primitive fiber and Guanidinium hydrochloride combination as the DNA bond material, thus preparation and a large amount of high purity DNA of purifying at short notice.

Background technology

Plasmid DNA is one of material the most basic in the molecular biology.Therefore, the preparation of the plasmid DNA of purifying is a technology the most frequently used in the molecular biology research.The manual preparation method of known many plasmid DNA in this area, and in many laboratories, be used now.Yet these manual methods are too consuming time, and unreliable.On the other hand, the laboratory of funds affluence is bought DNA and is prepared test kit, Qiagen for example, Germany and Promega, those that U.S.A. sells.Usually, but these product utilizations the advantage of plasmid DNA binding silica gel.

Volgelstein and Gillespie in their article, disclose a kind of from sepharose the method (1977) of DNA isolation.According to this method, DNA is combined on the flint glass, and this glass form is big flint glass particle, medium flint glass particle or flint glass powder, and also DNA can identify in NaI solution with combining of flint glass.Product based on this principle puts goods on the market, and its brand name is glass milk (glassmilk), makes that DNA isolation becomes easy from sepharose.As for the DNA separating effect, find that the flint glass powder has the most effective DNA binding ability, and medium flint glass particle shows that the DNA binding ability significantly descends.The poorest is big flint glass particle.In other words, along with the increase of flint glass granular size, the DNA separating effect reduces.

In addition, developed other material, comprised big borosilicate glass particle, glass fiber filter and Bio-Glas can be used to DNA isolation according to their binding ability.Yet these materials also have their weakness.For example, big borosilicate glass particle is the same with big flint glass, can not be effectively in conjunction with DNA.The DNA of glass fiber filter is in conjunction with very weak, therefore the DNA for preparing with small volume very little so that can not use.For Bio-Glas, obtain remarkable DNA bonding force, but consuming time very long in conjunction with DNA.When will dna degradation taking place when Bio-Glas reclaims DNA.In addition, the rate of recovery is too low, so its amount is done quantitative analysis inadequately.

Certainly, silica gel is very effective as the flint glass powder in the DNA combination.Yet it has problem on technological operation and DNA goods output.In any case silica gel has been used for DNA preparative column test kit and now by some companies (for example Qiagen and Promega) commercialization.Because product adopts the post mode of crossing based on gravity flowage, so its DNA preparation needs for a long time.In addition, the paste resin of use will be removed from DNA with the additional purification program.In addition, the dna solution that adopts existing DNA to prepare the test kit acquisition is not have endonuclease fully, and therefore self degraded can take place in standing storage.Another prominent question of currently available products is that they can not guarantee there is not albumen impurity (for example intracellular toxin) is present among the DNA of preparation.If in the dna solution of preparation, contain intracellular toxin, when being transfected into mammalian cell, dna vector can cause cytotoxicity, cause necrocytosis and cellullar immunologic response.Therefore need purer DNA goods to be used for the animal transfection.The preparation animal experiment is very expensive with the commercialization reagent of DNA.Therefore, prepare the high purity plasmid DNA with the transfection animal to being used to, but not expensive reagent there are strong needs.

The example of DNA purifying comprises and separates the radiolabeled probe, purified pcr product, and from the DNA prepared product, remove RNA and albumen impurity for example endonuclease and intracellular toxin.In addition, the DNA purifying can be applicable to cell proliferation test.For example, when the cell centrifugation sedimentation that will grow is added in the DNA preparative column, do not passed through post in containing isotopic nutrient solution by the isotropic substance of cellular uptake; Have only the isotropic substance of cellular uptake to be caught by post.Therefore, but need not any other expensive instrument the isotropic substance of quantitative assay cellular uptake just.

The disclosure of invention

Remember above-mentioned background, the present inventor has repeated the thoroughgoing and painstaking and extensive studies to DNA preparation and purifying, causes finding that borosilicate and Guanidinium hydrochloride combination have strong DNA binding ability.Basis of the present invention is the various advantages of borosilicate.Borosilicate is an inert on chemistry and physics, and is stable in weak acid and weak base.Its 1.0 micron particle have shown remarkable moisture absorption effectiveness.In addition, borosilicate has the good ability of removing impurity, even it can be by microbial contamination in long-time yet, even and loading capacity is little also can provide accurate analytical results.By with silica gel relatively confirmed the present invention superior on the DNA binding ability.The DNA binding ability of having found Guanidinium hydrochloride and borosilicate mixture than the high 2-4 of silica gel doubly.The present invention is achieved as the resin of a kind of DNA preparation and purification kit by using borosilicate and Guanidinium hydrochloride mixture.

Therefore, an object of the present invention is to overcome the problems referred to above that prior art is run into, and be provided for the post test kit of plasmid DNA preparation and purifying, with its can be easy at short notice a large amount of high purity DNA materials of acquisition.

Another object of the present invention provides this method that is used for the post test kit of plasmid DNA preparation and purifying of preparation.

Another object of the present invention provides a kind of DNA preparation and purification process, this method can be applicable to purify DNA from sepharose or polyacrylamide gel, the purifying radiolabeled probe, preparation PCR product, the plasmid DNA of purifying preparation, the preparation genomic dna, preparation RNA, and intracellular isotropic substance is gone in picked-up carry out quantitatively.

In the present invention, made and be used for the borosilicate glass mocrofibers dimension multilayer column that miniature, medium-sized and large-scale plasmid DNA prepares test kit, and the borosilicate glass mocrofibers dimension film/double-deck post of particle that is used for the DNA preparation.Use this test kit, prepared the high purity plasmid DNA, and be used for different tests, comprise the base sequencing analysis of the PCR product that cell, the order-checking of 35S dATP base and automatization base sequencing analysis, preparation are gone in the DNA transfection and the transduction of purifying and the DNA of expression and purification, thereby confirmed that DNA of the present invention prepares the superiority of test kit.

The accompanying drawing summary

Fig. 1 a is an exploded view, shows borosilicate glass mocrofibers dimension micro-column test kit of the present invention;

Fig. 1 b is a viewgraph of cross-section, shows the confined state of the post test kit of Fig. 1 a;

Fig. 2 is a histogram, has wherein drawn the plasmid DNA goods output with respect to borosilicate glass mocrofibers dimension film thickness.

Fig. 3 a is an exploded view, shows borosilicate glass mocrofibers dimension stake test kit of the present invention;

Fig. 3 b is a viewgraph of cross-section, shows the confined state of the post test kit of Fig. 3 a;

Fig. 4 a is an exploded view, shows glass microfiber film/particle intermediate column test kit;

Fig. 4 b is a viewgraph of cross-section, shows the confined state of the post test kit of Fig. 4 a;

Fig. 5 is a histogram, has wherein drawn the plasmid DNA goods output with respect to the glass microfiber film/particle bilayer thickness of intermediate column test kit.

Fig. 6 is a histogram, has wherein drawn the plasmid DNA goods output with respect to borosilicate and silicate glass primitive fiber filter membrane thickness;

Fig. 7 is the photo that digests and take behind electrophoresis on the sepharose with Restriction Enzyme by the plasmid DNA that post test kit of the present invention prepares;

Fig. 8 is the 35S dATP order-checking autography figure by the DNA of post test kit preparation of the present invention;

Fig. 9 a is a Photomicrograph, wherein expresses by the beta-galactosidase enzymes that dyeing identifies with conventional post test kit plasmid transfection cell preparation, that contain beta-galactosidase gene;

Fig. 9 b is a Photomicrograph, wherein uses post test kit of the present invention to prepare, and the cell that contains the plasmid transfection of beta-galactosidase gene is expressed by the beta-galactosidase enzymes that dyeing identifies;

Figure 10 is that automatic sequencing is analyzed photo, from obtaining by the pGEM 7Z of post test kit of the present invention with the preparation of T7 primer;

Figure 11 is that automatic sequencing is analyzed photo, from obtaining by the pGEM 7Z of post test kit of the present invention with the preparation of SP7 primer;

Figure 12 uses the plasmid DNA for preparing by post test kit of the present invention as template, the PCR product of acquisition, the photo behind electrophoresis on the sepharose;

To be plasmid DNA handle but without Ethanol Treatment with RNase Figure 13, and by post test kit of the present invention, the photo that obtains with rear electrophoresis; And

Figure 14 is the DNA band that downcuts from sepharose, by post test kit purifying of the present invention, and the photo behind the electrophoresis.

Implement optimal mode of the present invention

In the present invention, provide different DNA preparation and purification kits according to preparative capacibility.For this purpose, made the post that contains borosilicate glass mocrofibers dimension film or glass microfiber film and granular mixture.For example, made the borosilicate glass mocrofibers dimension multilayer column that is used for the miniature and large-scale preparation of DNA, and the borosilicate glass mocrofibers dimension matrix post that is used for the medium-sized preparation of DNA.With these post test kits, but high yield prepares the high purity plasmid DNA.Because post test kit of the present invention is very outstanding in conjunction with DNA, they can not only be used to prepare bacteria plasmid DNA, can also be from different pollutents or substrate material purify DNA.For example, the plasmid DNA that obtains with ordinary method can further be passed through post test kit purifying of the present invention.In addition, this post test kit can be applicable to purify DNA from sepharose and polyacrylamide gel.For example, behind electrophoresis on the sepharose, downcut required DNA band at them, and obtain by post test kit of the present invention from gel for the PCR product.Certainly, the purifying ability of post test kit of the present invention also is effective to the DNA band in the polyacrylamide gel.

The high quality DNA prepared product that obtains by post test kit of the present invention is very useful in senior molecular biology research, comprise gene clone, be transfected into mammalian cell and transduction, the base sequencing analysis, PCR, radioactive mark DNA's probe quantitative carries out cell proliferation biological test etc. with the radioactive mark DNA.

The following example is to be used for clearer those skilled in the art being illustrated principle of the present invention and practice.Same, they are not meaned and have limited the present invention, some preferences just are described.

The embodiment I

The manufacturing of GF post and the purifying of plasmid DNA

Experimental embodiment 1: be used for the manufacturing and uses thereof of the GF multilayer film post of the miniature preparation test kit of DNA

As shown in Figure 1a, at first, borosilicate glass mocrofibers is tieed up the circle that film is cut into 8 millimeters of diameters, and to be stacked on diameter be in 13 millimeters, dark 31 millimeters rotation micro-column 11, form themultilayered structure 13 of high 2-10 millimeter.Then, thering 12 of 7.5 millimeters of diameters is inserted in these posts, with fixing multilayer GF structure, then with this subassembly steam sterilizing.In order to use, the GF post of making is inserted in the collection tube 10.Fig. 1 b has shown the assembling situation ofpost 11 incollection tube 10.

In order to detect the suitableeest thickness of the glass microfiber multilayer film that can obtain highest purity maximum DNA, prepared different posts, its multilayer film respectively differs 2 millimeters.Use these posts to prepare plasmid DNA, the results are described in Fig. 2.Seen in this figure, when 8 millimeters of multilayer thickness, can obtain maximum (200 microgram).10 millimeters or be thicker than 10 millimeters film thickness DNA output is further improved.

Experimental embodiment 2: be used for the manufacturing and uses thereof of the GF multilayer film post of the large-scale preparation test kit of DNA

With having made the glass microfiber multilayer film post that is used for the large-scale preparation test kit of DNA withexperimental embodiment 1 similarity method.Shown in Fig. 3 a, at dark 86 millimeters, about 1 gram of packing in thehuge pillar 31 that diameter is 22 millimeters is cut into the borosilicate glassmocrofibers dimension film 33 of 22 millimeters circles of diameter, and circular membrane is fixing with cross pattern ring 32.In order to use, with 50 milliliters of tapered tubes as collection tube 30.Fig. 3 b shows the assembling situation of big GFpost 31 incollection tube 30.

Use this huge pillar, can from 250 milliliters of intestinal bacteria nutrient solutions, obtain about 1.2ml plasmid DNA.

Experimental embodiment 3: be used for the manufacturing and uses thereof of the GF film/particle post of miniature and medium-sized test kit

Borosilicate glass mocrofibers dimension mixed in mixing tank with the lmM EDTA solution that is dissolved in pH7.5,10mM Tris/HCl obtain the glass microfiber particle suspension.Shown in Fig. 4 a,, in the polyacrylamide post that diameter is 15 millimeters, theglass microfiber film 24 of 8 millimeters of diameters is piled up paramount 2 millimeters, the glass microfiber particle suspension of packing into therein then high 80 millimeters.This content to form the double-deck post of glass microfiber film/particle, is fixed steam sterilizing with the glass ring of 8 millimeters of diameters with the centrifugal settling of pendulum roller whizzer then.In order to use, this post is connected an Effendorfpipe 21,place collection tube 20 then.Fig. 4 b shows the assembling situation of this post.

Detected the plasmid DNA goods output with respect to the double-deck post degree of depth of glass microfiber film/particle, the result provides in Fig. 5.As can be seen, when the degree of depth is 8 millimeters, obtain maximum production.

With paper knife borosilicate glass mocrofibers is tieed up film and be cut into 3 millimeters of diameters or littler particle.These resins are packed in the micro-column with about 0.1g, with the 0.42g intermediate column of packing into, add the 4.2M Guanidinium hydrochloride then on post, centrifugal settling obtains active pillars.Micro-column can prepare plasmid DNA with about 100 microgram amounts, and intermediate column can prepare the plasmid DNA of about 200 micrograms.In the table 1, shown the output and the purity of the plasmid DNA goods that when using these posts, obtain hereinafter.

Table 1 plasmid DNA purity and goods output

Post	The e. coli jm109 nutrient solution	????OD260/OD280	Output
				GF matrix micro-column	10 milliliters	????1.7-1.9	About 100 micrograms
GF matrixintermediate column	100 milliliters	????1.7-1.9	About 200 micrograms

Experimental embodiment 4: the DNA goods output of using borosilicate GF post and preparing with silicate GF post

In order to detect the difference of borosilicate and silicate adsorptive power, borosilicate glass mocrofibers is tieed up the DNA goods output of post and the comparison of silicate glass microfiber column.As this post of manufacturing among theexperimental embodiment 1, and be used for preparing plasmid DNA from the intestinal bacteria nutrient solution.Analyze the output and the purity of the plasmid DNA that obtains, the results are described in Fig. 6.As seen in Fig., low about 20% of the DNA goods rate ratio borosilicate glass mocrofibers of silicate glass primitive fiber dimension post.Spectrophotometric determination is approximately 1.5 (OD with the plasmid DNA purity that the silicate glass microfiber column obtains₂₆₀/ OD₂₈₀), and tie up the plasmid DNA purity of post preparation between 1.7 to 1.8 scopes with borosilicate glass mocrofibers.

Experimental embodiment 5: DNA goods output and the purity using silicagel column and prepare with the double-deck post of silica gel/silicate GF

As shown inexperimental embodiment 3, every borosilicate glass mocrofibers that all forms 2 mm thick is tieed up film/granular layer in 5 intermediate columns, forms the layer of silica gel of 6 mm thick then thereon.Help by these posts has prepared plasmid DNA, and has analyzed its purity and output as experimental embodiment 2.The result provides in following table 2, show on silica gel to add borosilicate glass mocrofibers dimension layer, with single with silica gel relatively, remarkable output and the purity that improves the DNA goods.

In the following example, all plasmid DNA of use prepare by the silicate glass microfiber column.The purity of table 2 plasmid DNA and goods output

Post	Intestinal bacteria JMi09 nutrient solution	????OD260/OD280	Output
				Silica gel
	10 milliliters	????1.7-1.9	About 100 micrograms
				Silica gel/borosilicate GF	100 milliliters	????1.7-1.9	About 200 micrograms

The embodiment II

The preparation of plasmid DNA

Use GF micro-column test kit, GF intermediate column or the GF stake test kit of the manufacturing of embodiment I to prepare plasmid DNA.At first, cultivate the coli strain that has certain plasmid in meat soup, centrifugation medium obtains (granular) precipitation.This cell precipitation is suspended with buffer A (50mM Tris, 10mM EDTA pH8.0,100 micrograms/ml RNaseA), use buffer B (20mM NaOH, 1.0%SDS (w/v)) cracking, and (the 3.2mM Potassium ethanoate pH5.0) neutralizes with damping fluid C.Centrifugal should in and lysate, supernatant liquor is added to (this post before activated with Guanidinium hydrochloride) on the GF post test kit, DNA is attached on the post.After removing nuclease, the DNA of washing and elution of bound.In order to remove nuclease, used the 4.2M Guanidinium hydrochloride.As washings, used 10mMTris/HCl (pH7.5), 50mMNaCl, 0.1mM EDTA, 70% ethanol.

The embodiment III

Molecular biology test and analysis

Experimental embodiment 1: digest with Restriction Enzyme

With the plasmid DNA for preparing in Restriction Enzyme EcoR I and the Hind III Processing Example II, and on sepharose with complete plasmid DNA electrophoresis.As seen in Figure 7, the plasmid DNA of preparation is clearly by endonuclease digestion.

Experimental embodiment 2:³⁵S dATP sequencing analysis

(for example the test kit sold of United StatesBiochemical is called Sequenase to make the 10 microgram plasmid DNA that prepare in the embodiment II and sequencing kit^TM2.0 version) reaction is used³⁵S dATP is as radio-labeling, and 10 microgram plasmid DNA by the preparation of Qiagen test kit in contrast.Read sequencing result by radioactive automatic developing from sequencing gel.As seen in fig. 8, the autography band of the plasmid DNA by post preparation of the present invention clearly separates, and fails effectively separately by the band that the Qiagen test kit obtains.

Experimental embodiment 3: cell transfecting and transduction

To go into mammalian cell by the plasmid DNA transfection and the transduction of post test kit of the present invention preparation, and the plasmid DNA by the preparation of Qiagen test kit is with comparing.Shown in Fig. 9 b, the plasmid DNA by post test kit of the present invention preparation obtains effective expression in cell as identifying with the protein that dyes.On the contrary, contrast DNA shows faint expression efficiency, shown in Fig. 9 a.

Experimental embodiment 4: automatic base sequencing analysis

Use ABI PRIM^TMThe terminal cycle sequencing of dyestuff reads reaction kit (commerce can derive from PerkinElmer) and has carried out automatic base sequencing analysis.Will be by the 0.4 microgram template DNA and the 3pM primer of post of the present invention preparation, and 8 microlitre ends read reaction mixture, and adjust tocumulative volume 20 microlitres.This solution carries out 25 and takes turns reaction, and each wheel is included in thermal cycler, and (for example Bio Rad sells, and is called Gene Cycler^TM) in 96 ℃/30 minutes, 50 ℃/15 seconds and 60 ℃/4 minutes.With the DNA product that ethanol sedimentation extends, dry in SpeedVac, and in automatic sequencer (for example Applied Biosystem sells), analyze.Make template with wherein having inserted the segmental plasmid pGEM7z of long 1.2kbDNA.T7 or SP6 primer have been used.

The purifying of experimental embodiment 5:PCR reaction and PCR product

Use has been carried out the PCR reaction by the CMV-TNF plasmid DNA of embodiment I post preparation as template.To contain 20 nanogram CMV-TNF plasmids, the 1mM dNTP of 2 microlitres, the 10x reaction buffer of 5 microlitres, the 2UTag polysaccharase, have adopted primer or promotor distally that 95 ℃ of preheatings ofreaction mixture 2 minutes of adopted primer associating TNF antisense primer are arranged with the promotor nearside, carry out 30 then and take turns reaction, the every wheel comprises 95 ℃/1 minute, 55 ℃/1 minute and 72 ℃/1 minute, and at last 72 ℃ ofextensions 10 minutes.Reaction product is divided into two five equilibriums.One five equilibrium mixes with isopyknic 4.2M Guanidinium hydrochloride, also uses the WA solution washing by the minim DNA purification column.The DNA of this purifying of electrophoresis on sepharose, and another five equilibrium is in contrast.Electrophoresis result is shown in Figure 12.As this figure finding, in PCR product swimming lane, all primer bands have been found with minim DNA purification column purifying.

Experimental embodiment 6:DNA purifying

From the nutrient solution of three 250 ml volumes, use the GF matrix stake of embodiment I manufacturing to prepare plasmid DNA.At first, first culture has been used the typical method of using WA solution.Use ethanol sedimentation, obtained the DNA of second culture, be dissolved in then in the 500 microlitre deionized waters without the WA solution washing.For last culture, as second culture, obtained DNA, be divided into the volume of 250 microlitres.In each five equilibrium, add 20 microlitre RNaseA (3mg/ml), 37 ℃ of reactions 30 minutes, 65 ℃ of deactivations 10 minutes, and by micro-post.Make DNA product electrophoresis on sepharose of acquisition like this, as shown in figure 13.Data provide in the following Table 3.As illustrated in table 3 data, when without Ethanol Treatment, can obtain a large amount of plasmid DNA, but have RNA to pollute.On the other hand, handle and cross the plasmid DNA of column purification acquisition with RNaseA and do not have RNA impurity fully, and show DNA purity (OD₂₈₀/ OD₂₆₀) bring up to 1.90 from 1.72.Therefore, the present invention can overcome conventional DNA preparation method's critical defect, that is, RNA pollutes and low DNA output and purity.Table 3

	????OD280/OD260	Concentration	Total DNA
				The WA washing	????1.44	????7.60	760 micrograms
Not washing	????1.68	????10.80	1.08 milligram
				Washing/column purification not	????1.90	????11.75	1.175 milligram

Experimental embodiment 7: DNA isolation on sepharose or the polyacrylamide gel

Dna solution is gone up electrophoresis at sepharose/TAE or TBE, and downcuts a DNA band from glue, places dialysis tubing, the TAE solution of packing into therein then, and carry out electrophoresis DNA isolation from the gel.In TAE solution, add isopyknic 4.2M Guanidinium hydrochloride, and be added on the GF trace post of the present invention.Column spinner is also used WA solution (10mM Tris/HCl pH7.5,30mM EDTA, 70% ethanol) washing.Then with 50 microlitre deionized waters rotation eluted dna.DNA identifies by electrophoresis on sepharose and analyzes, as shown in figure 14.

Analytical results is summarized in hereinafter table 4.According to these results, the present invention has following advantage.The first, post test kit of the present invention makes simple and rapid preparation plasmid DNA become possibility.The second, post test kit of the present invention has shown high DNA goods output.With conventional column spinner test kit relatively, the DNA that GF micro-column test kit of the present invention can the amount of obtaining high about 50 to 100 times (about 200 micrograms).Use GF intermediate column test kit of the present invention, can obtain the DNA of as many as 400 micrograms, and GF stake test kit obtains the 2.2mg plasmid DNA.The 3rd, the DNA purity (OD of the plasmid DNA that obtains by post test kit of the present invention₂₆₀/ OD₂₈₀) be 1.7 to 1.9 with the spectrophotometric determination scope.The condition of plasmid DNA output that can effectively prepare the maximum of the plasmid DNA table 5 that sees below.At last, nuclease (it often appears in the colibacillary DNA prepared product), and carbohydrate (it is the pollutent that comes across in the DNA prepared product of JM109 bacterial strain) in the DNA prepared product that obtains with post test kit of the present invention from undiscovered mistake.The plasmid DNA that table 4 prepares with GF intermediate column test kit

Reagent	The JM109 nutrient solution	Plasmid	OD260/OD280	Maximum production
					Chaotropic salt	????200ml	????pGEM?7Z	????1.7-1.8	The 50-100 microgram
Guanidinium hydrochloride	????200ml	????pGEM?7Z	????1.7-1.9	The 200-400 microgram

The plasmid DNA that table 5 prepares with GF post test kit

Test kit*	The JM109 nutrient solution	Plasmid	OD260/OD280	The GF resin	Output
						Small-sized	10ml	?pGEM?7Z	?1.7-1.8	?0.1g	About 70 micrograms
Medium-sized	100ml	?pGEM?7Z	?1.6-1.9	?0.42g	About 200 micrograms
						Large-scale	250ml	?pGEM?7Z	?1.7-1.9	?1-1.34g	?832μg-2.2mg

^*GF micro-column and intermediate column: matrix type; GF stake: multilayer type

Industrial applicability

Mentioned above as this paper, GF post test kit of the present invention is useful to a large amount of plasmid DNA of simple and rapid preparation from inoculum.This plasmid DNA purity is very high, therefore need not to be further purified just to can be applicable to various senior experiments, for example base sequencing analysis, transfection and transduction, clone etc.In addition, post test kit of the present invention can be used for purify DNA from sepharose or polyacrylamide gel, and its purifying ability also can be applicable to separate the radiolabeled probe, purified pcr product, from the DNA prepared product, remove RNA and protein impurities (for example endonuclease and intracellular toxin), and cell proliferation test.Therefore, the present invention is very valuable in biological medicine industry.

Though the present invention has made detailed description with reference to some preferences, be understandable that, can carry out different modifications within the spirit and scope of the present invention.The present invention require except that right those unrestricted.

Claims (13)

1. a glass microfiber multilayer column test kit that is used to prepare with purify DNA is characterized in that described test kit comprises a post, contains resin in this post, and described resin is formed by multiple glazing primitive fiber film.

2. a glass microfiber matrix post test kit that is used to prepare with purify DNA is characterized in that described test kit comprises a post, contains resin in this post, and described resin is formed by double glazing primitive fiber film and glass microfiber particle.

3. glass microfiber multilayer column test kit as claimed in claim 1 is characterized in that, described post comprises a miniature column spinner test tube; Be used in conjunction with DNA by the glass microfiber film form multi-layer resinous; One is fixed on ring or glass in the miniature column spinner test tube with the glass microfiber film; With the collection tube of a collection by the material of glass microfiber film.

4. glass microfiber matrix post test kit as claimed in claim 2 is characterized in that described post comprises an intermediate column test tube; Be used in conjunction with the glass microfiber film of DNA and the double-layer resin of glass microfiber granulometric composition; The Eppendorf tube of collection DNA by double-layer resin, the separable and 1.5ml that the intermediate column test tube is supporting; The collection tube of the material of double-layer resin is passed through in a collection, has settled described intermediate column and described 1.5ml Eppendorf tube in this collection tube; One is fixed on ring or glass in the described intermediate column test tube with double-layer resin.

5. glass microfiber multilayer column test kit as claimed in claim 1 is characterized in that described glass microfiber film is formed by the mixture of borosilicate or silicate and borosilicate.

6. glass microfiber multilayer column test kit as claimed in claim 1 is characterized in that, described glass microfiber multilayer column test tube has miniature, medium-sized or large-scale.

7. glass microfiber matrix post test kit as claimed in claim 2 is characterized in that described glass microfiber film and glass microfiber particle are formed by the mixture of borosilicate or silicate and borosilicate.

8. post test kit as claimed in claim 1 or 2 is characterized in that, described glass microfiber matrix post test tube has miniature, medium-sized or large-scale.

9. a method for preparing plasmid DNA is characterized in that, uses claim 1 described glass microfiber film multilayer column test kit or the described glass microfiber matrix of claim 2 post test kit.

10. the method for a DNA isolation from sepharose or polyacrylamide gel is characterized in that, uses claim 1 described glass microfiber film multilayer column test kit or the described glass microfiber matrix of claim 2 post test kit.

11. isotopic method in the quantitative analysis cell is characterized in that, uses claim 1 described glass microfiber film multilayer column test kit or the described glass microfiber matrix of claim 2 post test kit.

12. the method for a purify DNA is characterized in that, use claim 1 described glass microfiber film multilayer column test kit or the described glass microfiber matrix of claim 2 post test kit are removed RNA or the protein pollutant among the DNA.

13. a purifying radiolabeled probe method is characterized in that, uses claim 1 described glass microfiber film multilayer column test kit or the described glass microfiber matrix of claim 2 post test kit.

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