CN202246705U - Reagent box for extracting nucleic acid according to magnetic bead - micropore plate method - Google Patents
Reagent box for extracting nucleic acid according to magnetic bead - micropore plate method Download PDFInfo
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- CN202246705U CN202246705U CN201120348292XU CN201120348292U CN202246705U CN 202246705 U CN202246705 U CN 202246705U CN 201120348292X U CN201120348292X U CN 201120348292XU CN 201120348292 U CN201120348292 U CN 201120348292U CN 202246705 U CN202246705 U CN 202246705U
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- microwell plate
- magnetic
- nucleic acid
- magnetic bead
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Abstract
The utility model discloses a reagent box for extracting nucleic acid according to a magnetic bead -micropore plate method. The reagent box comprises a box body and a reagent, wherein the box body is composed of a detachable micropore plate (2) like an ELISA plate and a magnetic plate (1). The reagent comprises a magnetic bead, pyrolysis-combination liquid, cleaning solution A, cleaning solution B and eluent. The detachable micropore plate (2) is composed of micropores (4) and a pore frame (3). The micropores (4) are made of polystyrene, polyethylene or polypropylene, the bottoms of the micropores are in a U shape or a V shape, and the micropores (4) are of two forms: single pore and multi-linking pore. The shape and the dimension of the magnetic plate (1) are matched with the bottom of the detachable micropore plate (2). The reagent box can extract nucleic acid of a sample massively in one time and is simple to operate, low in cost and high in production efficiency.
Description
Technical field
The utility model relates to a kind of test kit that is used to extract nucleic acid, has been specifically related to a kind of magnetic bead-microwell plate method and has extracted the nucleic acid reagent box.
Background technology
Nucleic acid is one type of bioinformation macromolecular compound, from cell, is separated first from it, has experienced the history in more than 100 year, until eighties of last century after mid-term scientist nucleic acid construct and function have just been obtained the development of leap.Nucleic acid is divided into thymus nucleic acid and Yeast Nucleic Acid, and Nucleotide is the fundamental unit of nucleic acid.DNA and RNA have existence in the plant-animal kind, but a kind of virus only contains a kind of nucleic acid, DNA or RNA.The effect that can not be substituted that had of nucleic acid in whole molecular biology since molecule biology is founded, has been the main object of molecular biology research always.Therefore, the extraction of nucleic acid and separation and purification also are the indispensable links of all kinds of experiments of molecular biology.In clinical diagnosis,,, be used for the diagnosis and the tissue matching of disease then like separation and purification nucleic acid in serum or blood plasma, the tissue slice often from tissue.
Traditional chemical processes such as phenol-chloroform method are extracted nucleic acid; Though the nucleic acid yield that this method is extracted is high, purity is good, use hazardous and noxious substances such as phenol and chloroform; Operating process repeatedly the open pipe liquid feeding, centrifugal, abandon liquid; Frequent change centrifuge tube and suction are chewed, and algorithm is tediously long numerous and diverse, cause the extraction working efficiency low.
Development along with nanotechnology utilizes SiO
2The characteristic of the special under certain condition absorption nucleic acid of nanometer magnetic bead of (silicon-dioxide) parcel has been developed magnetic bead and has been extracted the purification of nucleic acid technology, and this technology has obtained using widely.Operation primary process is after nucleic acid and the magnetic bead thorough mixing in the sample makes nucleic acid be adsorbed onto magnetic bead; With magnetic bead under the effect in magnetic field and solution separating; After washing several times; With elutriant nucleic acid is separated with magnetic bead, by from liquid phase, separating in the magnetic field, liquid phase is the nucleic acid solution of purifying to magnetic bead again.So paramagnetic particle method extracts the physical property of this techniques make use magnetic bead of nucleic acid and extracts purification of nucleic acid; Do not use the poisonous and harmful chemical reagent; Operating process is simple, chews without frequent change centrifuge tube and suction, need not be centrifugal; Magnetic moment can accomplish, and nucleic acid yield and purity are superior to chemical processes such as phenol-chloroform method.The more traditional chemical processes such as phenol-chloroform method of paramagnetic particle method are revolutionary technical renovation.Disclose a kind of test kit of using nanometer magnetic beads for purifying nucleic acid like publication number CN101684138A, this test kit is made up of lysate, magnetic bead buffer solution, lavation buffer solution 1, lavation buffer solution 2, lavation buffer solution 3, elution buffer and balance liquid; It is fast, highly sensitive that nucleic acid speed is extracted in this invention, and have the potentiality of automatic updating than traditional nucleic acid purification process, makes the LSI of nucleic acid extraction, and homogeneity becomes feasible.But existing paramagnetic particle method extracts nucleic acid also exists following shortcoming: 1. owing in centrifuge tube, extract, the liquid feeding of need uncapping repeatedly, cover mixing, uncap again and abandon the liquid process, and when detection limit is tens increments up to a hundred, the extremely inconvenience of uncapping repeatedly.2. the molecular Biological Detection amount of samples is generally 1～5 microlitre, and conventional extracted amount is 50～100 microlitres, and the cost expensive with magnetic bead compares, and has bigger waste undoubtedly.
Summary of the invention
The purpose of the utility model is the deficiency to prior art, provides a kind of magnetic bead-microwell plate method simple to operate, that practice thrift cost to extract the nucleic acid reagent box.
To achieve these goals; The utility model takes following technology to solve bill: a kind of magnetic bead-microwell plate method is extracted the nucleic acid reagent box; Comprise box body and reagent; Box body comprises magnetic board and detachable microwell plate, and detachable microwell plate is made up of hole frame and micropore two portions, and micropore divides single hole or joins the hole more and is placed on the frame of hole; Reagent comprises magnetic bead, cracking-combination liquid, washings A, washings B and elutriant.
The manufacturing materials of above-mentioned micropore is PS, Vilaterm or Vestolen PP 7052.
Be " U " shape or " V " shape at the bottom of the hole of said micropore, the volume of micropore is more than 400 microlitres.
Said magnetic board is a rectangle Nd-Fe-B strong magnetic piece, and shape, size and detachable microwell plate bottom are adaptive.
Said magnetic bead is SiO
2The ferric oxide nanometer particle of parcel; Said cracking-combination liquid is the guanidinium isothiocyanate Digestive system, and said washings A is the 75% absolute ethyl alcohol aqueous solution, and said washings B is an absolute ethyl alcohol; Said elutriant is that Dian Dao is Shuaied ≧ pure water of 18 little siemenss, i.e. ultrapure water or distilled water.
The sample that said test kit extracts nucleic acid is solid, liquid or solid-liquid miscellany.
All reagent are working fluid in the mentioned reagent box, can directly use, and magnetic bead liquid and cracking-combination liquid fully shakes up before using; Test kit stores under normal temperature condition and transports, and magnetic bead liquid and magnetic sheet are deposited distance at least more than the 10cm, are valid for one year; Reagent of the present invention and apparatus are used, and do not advise using with other reagent or apparatus.
Use the method for the utility model test kit extraction nucleic acid following:
1. sample and cracking-combine liquid is put into centrifuge tube fully after the effect, under 4000～10000 rev/mins rotating speed, centrifugal 5～15 minutes;
2. get the micropore that microwell plate is put in supernatant 50～200 microlitres/hole, the corresponding hole of record sample number, and with 8 passage micropipets to micropore adding magnetic bead 5～10 microlitres/hole;
3. microwell plate is placed on abundant mixing in the micro-vortex oscillation device; Left standstill 5～10 minutes; Middle and last each mixing 1～2 time, with buckling on the magnetic board at the bottom of the microwell plate plate, magnetic is picked up magnetic board and microwell plate after 10～30 seconds together then; Get rid of liquid in the clear opening, the mouth of pipe downwards on thieving paper tapping to blot residual liquid;
4. separate microwell plate and magnetic board earlier; Add washings A with 8 passage micropipets to micropore then, 50～200 microlitres/hole are placed on microwell plate that vibration fully suspends magnetic bead in the micro-vortex oscillation device; Again with buckling on the magnetic board at the bottom of the microwell plate plate; Magnetic is picked up magnetic board and microwell plate after 10～30 seconds together, gets rid of liquid in the clear opening, then the mouth of pipe downwards on thieving paper tapping to blot residual liquid; This step 3 of repetitive operation～5 time;
5. separate microwell plate and magnetic board once more; Add washings B with 8 passage micropipets to micropore then, 50～200 microlitres/hole are placed on microwell plate that vibration fully suspends magnetic bead in the micro-vortex oscillation device; Again with buckling on the magnetic board at the bottom of the microwell plate plate; Magnetic is picked up magnetic board and microwell plate after 10～30 seconds together, gets rid of liquid in the clear opening, then the mouth of pipe downwards on thieving paper tapping to blot residual liquid;
6. microwell plate is moved into that 45～65 ℃ of dryings added elutriant with 8 passage micropipets to micropore after 10～20 minutes in the loft drier, 30～200 microlitres/hole are placed on microwell plate that vibration fully suspends magnetic bead in the micro-vortex oscillation device;
7. microwell plate is moved in the thermostat container 60～65 ℃ of effects 10～20 minutes, during vibration mixing 2～3 times, with buckling on the magnetic board at the bottom of the microwell plate plate, magnetic 10～30 seconds, supernatant are the nucleic acid that extracts purifying at last.
Compared with prior art, the advantage of the utility model:
1. micropore is fixed in the grillage, and multichannel pipettor easy to use adds, gets liquid and abandons liquid, overcome former technology by pipe uncap repeatedly liquid feeding with abandon loaded down with trivial details operation such as liquid, nucleic acid that can disposable extraction sample in enormous quantities, working efficiency is more than 10 times of former technology.This test kit also provides cover nucleic acid extraction apparatus a---box body when extracting reagent providing, and is made up of with the adaptive magnetic board of microwell plate a detachable microwell plate that is similar to enzyme plate and one; Magnetic bead and nucleic acid absorption, washing, wash-out, magnetic etc. all carry out in the hole, are similar to the ELISA operating process, need not pursue and manage the liquid feeding of uncapping repeatedly, abandon loaded down with trivial details operations such as liquid, and reagent dosage is saved over half than the centrifuge tube mode.
2. consumptive material is cheaply economical, and its nucleic acid extraction cost is that machine is with 1/10 of deep-well plates method.
3. reagent consumption is few, and it is 1/2 of traditional method and deep-well plates method consumption that magnetic bead application of sample amount is merely reagent such as 5ul.The effect of magnetic board provides the magnetic effect, is getting rid of when abandoning in the hole liquid, and magnetic board and microwell plate are close to, and hold magnetic bead, prevent that magnetic bead from throwing away with liquid.
Description of drawings
Fig. 1 is the synoptic diagram of the utility model one embodiment test kit.
Fig. 2 is the magnetic board synoptic diagram of the utility model.
Fig. 3 is the synoptic diagram of the detachable 8 hole micropores of the utility model.
Fig. 4 is the synoptic diagram of the micropore plate hole frame of the utility model.
Fig. 5 is the synoptic diagram of 8 * 12 specification microwell plates of the utility model.
Reference numeral: magnetic board 1, microwell plate 2, hole frame 3, micropore 4.
Embodiment
Below in conjunction with accompanying drawing the utility model is further specified.
Embodiment 1:
Shown in accompanying drawing, use PS to make the micropore 4 that volume is 8 holes that take the shape of the letter U at the bottom of 400 microlitres, the hole, then micropore 4 is put into corresponding hole frame 3, form detachable microwell plate 2; The detachable microwell plate 2 that assembles is kowtowed on the adaptive magnetic board 1 in shape, size and detachable microwell plate 2 bottoms, micropore 4 bottoms and magnetic board 1 are close together, the magnetic bead-microwell plate method that can obtain the utility model is extracted the nucleic acid reagent box.
Embodiment 2:
Shown in accompanying drawing, use Vestolen PP 7052 to make the micropore 4 that volume is 8 holes that take the shape of the letter U at the bottom of 500 microlitres, the hole, then micropore 4 is put into corresponding hole frame 3, form detachable microwell plate 2; The detachable microwell plate 2 that assembles is kowtowed on the adaptive magnetic board 1 in shape, size and detachable microwell plate 2 bottoms, micropore 4 bottoms and magnetic board 1 are close together, the magnetic bead-microwell plate method that can obtain the utility model is extracted the nucleic acid reagent box.
Embodiment 3:
Using Vilaterm to make volume is to be the single hole micropore 4 of V-arrangement at the bottom of 550 microlitres, the hole, then a plurality of single hole micropores 4 is put into the hole frame 3 of respective number, forms detachable microwell plate 2; The detachable microwell plate 2 that assembles is kowtowed on the adaptive magnetic board 1 in shape, size and detachable microwell plate 2 bottoms, micropore 4 bottoms and magnetic board 1 are close together, the magnetic bead-microwell plate method that can obtain the utility model is extracted the nucleic acid reagent box.
Embodiment 4:
The step of extracting the DNA in nucleic acid reagent box extraction serum/whole blood sample with magnetic bead-microwell plate method of the foregoing description 1 is following:
1. the guanidinium isothiocyanate Digestive system (V/V) that in centrifuge tube, adds serum/whole blood and 3～5 times of amounts, fully after the mixing effect, under 4000 rev/mins rotating speed centrifugal 5 minutes;
2. get supernatant 50 microlitres/hole and put into microwell plate 2 apertures, the corresponding hole of record sample number, and with 8 passage micropipets adding magnetic bead, 5 microlitres/hole;
3. with microwell plate 2 abundant mixing in micro-vortex oscillation device; Left standstill 5 minutes; Middle and last each mixing 1 time, with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates, magnetic is picked up magnetic board 1 and microwell plate 2 after 10 seconds together then; Get rid of liquid in the clear opening, the mouth of pipe downwards on thieving paper tapping to blot residual liquid;
4. separate microwell plate 2 and magnetic board 1 earlier; Add the 75% absolute ethyl alcohol aqueous solution with 8 passage micropipets to microwell plate then, 50 microlitres/hole are placed in the micro-vortex oscillation device vibration with microwell plate 2 magnetic bead are fully suspended; Again with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates; Magnetic is picked up magnetic board 1 and microwell plate 2 after 10 seconds together, gets rid of liquid in the clear opening, then the mouth of pipe downwards on thieving paper tapping to blot residual liquid; This step 3 of repetitive operation time;
5. separate microwell plate 2 and magnetic board 1 once more; Add absolute ethyl alcohols with 8 passage micropipets to microwell plate 2 then, 50 microlitres/hole are placed on microwell plate 2 that vibration fully suspends magnetic bead in the micro-vortex oscillation device; Again with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates; Magnetic is picked up magnetic board 1 and microwell plate 2 after 10 seconds together, gets rid of liquid in the clear opening, then the mouth of pipe downwards on thieving paper tapping to blot residual liquid;
6. microwell plate 2 was moved in the loft drier 45 ℃ of dryings after 10 minutes, add Dian Dao with 8 passage micropipets to microwell plate 2 and Shuai ≧ ultrapure water of 18 little siemenss, 30 microlitres/hole are placed on microwell plate 2 to vibrate in the micro-vortex oscillation device magnetic bead are fully suspended;
7. microwell plate 2 is moved in the thermostat containers 60 ℃ of effects 10 minutes, during vibration mixing 2 times, with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates, magnetic 10 seconds, supernatant are the DNA acid of extracting purifying at last.
The result of embodiment 4: obtain purity 1.8＜OD260/ OD280＜2.0 of aqueous dna, volume >=30ul.
Embodiment 5:
The step of extracting the DNA in the nucleic acid reagent box extraction tissue homogenate sample with magnetic bead-microwell plate method of the foregoing description 2 is following:
1. the guanidinium isothiocyanate Digestive system (V/V) that in centrifuge tube, adds tissue homogenate and 3～5 times of amounts, fully after the mixing effect, under 7500 rev/mins rotating speed centrifugal 10 minutes;
2. get supernatant 100 microlitres/hole and put into microwell plate 2 apertures, the corresponding hole of record sample number, and with 8 passage micropipets adding magnetic bead, 8 microlitres/hole;
3. with microwell plate 2 abundant mixing in micro-vortex oscillation device; Left standstill 7 minutes; Middle and last each mixing 2 times, with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates, magnetic is picked up magnetic board 1 and microwell plate 2 after 20 seconds together then; Get rid of liquid in the clear opening, the mouth of pipe downwards on thieving paper tapping to blot residual liquid;
4. separate microwell plate 2 and magnetic board 1 earlier; Add the 75% absolute ethyl alcohol aqueous solution with 8 passage micropipets to microwell plate 2 then, 120 microlitres/hole are placed on microwell plate 2 that vibration fully suspends magnetic bead in the micro-vortex oscillation device; Again with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates; Magnetic is picked up magnetic board 1 and microwell plate 2 after 20 seconds together, gets rid of liquid in the clear opening, then the mouth of pipe downwards on thieving paper tapping to blot residual liquid; This step 4 of repetitive operation time;
5. separate microwell plate 2 and magnetic board 1 once more; Add absolute ethyl alcohols with 8 passage micropipets to microwell plate 2 then, 150 microlitres/hole are placed on microwell plate 2 that vibration fully suspends magnetic bead in the micro-vortex oscillation device; Again with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates; Magnetic is picked up magnetic board 1 and microwell plate 2 after 20 seconds together, gets rid of liquid in the clear opening, then the mouth of pipe downwards on thieving paper tapping to blot residual liquid;
6. microwell plate 2 was moved in the loft drier 55 ℃ of dryings after 15 minutes, add Dian Dao with 8 passage micropipets to microwell plate 2 and Shuai ≧ distilled water of 18 little siemenss, 100 microlitres/hole are placed on microwell plate to vibrate in the micro-vortex oscillation device magnetic bead are fully suspended;
7. microwell plate 2 is moved in the thermostat containers 62 ℃ of effects 15 minutes, during vibration mixing 2 times, with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates, magnetic 20 seconds, supernatant are the DNA that extracts purifying at last.
The result of embodiment 5: obtain purity 1.8＜OD260/ OD280＜2.0 of aqueous dna, volume >=30ul.
Embodiment 6:
The step of extracting the DNA in the nucleic acid reagent box extraction faecal samples with magnetic bead-microwell plate method of the foregoing description 3 is following:
1. the guanidinium isothiocyanate Digestive system (V/V) that in centrifuge tube, adds faecal samples and 3～5 times of amounts, fully after the mixing effect, under 10000 rev/mins rotating speed centrifugal 15 minutes;
2. get supernatant 200 microlitres/hole and put into microwell plate 2 apertures, the corresponding hole of record sample number, and with 8 passage micropipets adding magnetic bead, 10 microlitres/hole;
3. with microwell plate 2 abundant mixing in micro-vortex oscillation device; Left standstill 10 minutes; Middle and last each mixing 2 times, with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates, magnetic is picked up magnetic board 1 and microwell plate 2 after 30 seconds together then; Get rid of liquid in the clear opening, the mouth of pipe downwards on thieving paper tapping to blot residual liquid;
4. separate microwell plate 2 and magnetic board 1 earlier; Add the 75% absolute ethyl alcohol aqueous solution with 8 passage micropipets to microwell plate 2 then, 200 microlitres/hole are placed on microwell plate 2 that vibration fully suspends magnetic bead in the micro-vortex oscillation device; Again with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates; Magnetic is picked up magnetic board 1 and microwell plate 2 after 30 seconds together, gets rid of liquid in the clear opening, then the mouth of pipe downwards on thieving paper tapping to blot residual liquid; This step 5 of repetitive operation time;
5. separate microwell plate 2 and magnetic board 1 once more; Add absolute ethyl alcohols with 8 passage micropipets to microwell plate 2 then, 200 microlitres/hole are placed on microwell plate 2 that vibration fully suspends magnetic bead in the micro-vortex oscillation device; Again with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates; Magnetic is picked up magnetic board 1 and microwell plate 2 after 30 seconds together, gets rid of liquid in the clear opening, then the mouth of pipe downwards on thieving paper tapping to blot residual liquid;
6. microwell plate 2 was moved in the loft drier 65 ℃ of dryings after 20 minutes, add Dian Dao with 8 passage micropipets to microwell plate 2 and Shuai ≧ ultrapure water of 18 little siemenss, 200 microlitres/hole are placed on microwell plate 2 to vibrate in the micro-vortex oscillation device magnetic bead are fully suspended;
7. microwell plate 2 is moved in the thermostat containers 65 ℃ of effects 20 minutes, during vibration mixing 3 times, with buckling on the magnetic board 1 at the bottom of microwell plate 2 plates, magnetic 30 seconds, supernatant are the DNA that extracts purifying at last.
The result of embodiment 6: obtain purity 1.8＜OD260/ OD280＜2.0 of aqueous dna, volume >=30ul.
Claims (3)
1. magnetic bead-microwell plate method is extracted the nucleic acid reagent box; Comprise box body; It is characterized in that: box body comprises magnetic board (1) and detachable microwell plate (2), and detachable microwell plate (2) is made up of hole frame (3) and micropore (4) two portions, micropore (1) branch single hole or join the hole more and be placed on the hole frame (3).
2. extract the nucleic acid reagent box according to the said magnetic bead of claim 2-microwell plate method, it is characterized in that: be " U " shape or " V " shape at the bottom of the hole of said micropore (4), the volume of micropore (4) is more than 400 microlitres.
3. extract the nucleic acid reagent box according to the said magnetic bead of claim 1-microwell plate method, it is characterized in that: said magnetic board (1) is a rectangle Nd-Fe-B strong magnetic piece, and shape, size and detachable microwell plate (2) bottom are adaptive.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201120348292XU CN202246705U (en) | 2011-09-16 | 2011-09-16 | Reagent box for extracting nucleic acid according to magnetic bead - micropore plate method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201120348292XU CN202246705U (en) | 2011-09-16 | 2011-09-16 | Reagent box for extracting nucleic acid according to magnetic bead - micropore plate method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN202246705U true CN202246705U (en) | 2012-05-30 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201120348292XU Expired - Fee Related CN202246705U (en) | 2011-09-16 | 2011-09-16 | Reagent box for extracting nucleic acid according to magnetic bead - micropore plate method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN202246705U (en) |
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2011
- 2011-09-16 CN CN201120348292XU patent/CN202246705U/en not_active Expired - Fee Related
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120530 Termination date: 20140916 |
|
| EXPY | Termination of patent right or utility model |