CN101705225B - High-efficiency separation method for lower molecular multiplex PCR-amplified fragments - Google Patents
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Abstract
The invention provides a high-efficiency separation method for lower molecular multiplex PCR-amplified fragments, which comprises: firstly, purifying lower molecular multiplex PCR-amplified fragments by using a magnetic bead purification method; and secondly, separating the lower molecular multiplex PCR-amplified fragments by using a capillary electrophoresis method. The method combining a multiplex PCR method and capillary electrophoresis can quickly screen transgenic plants. The multiplex PCR has high template-saving, reagent-saving and time-saving performance; when used to process the products of the multiplex PCR, the magnetic bead method can effectively remove the impurities from a PCR reaction system to make the PCR reaction system more suitable for performing capillary electrophoresis; and the capillary electrophoresis has the characteristics of low detection limit, high sensitivity and short detection time. The method can be safely used, successfully separate and identify the lower molecular multiplex PCR-amplified fragments, keep the detection time within 10.6 minutes and be widely used in fields such as food sanitation quarantine and clinical examination.
Description
Technical field
The present invention relates to a kind of method, specifically, relate to a kind of method.The present invention can success lower molecular multiplex PCR-amplified fragments is carried out high efficiency separation, multiple PCR products can better separate it with capillary electrophoresis through behind the magnetic beads for purifying.
Background technology
The security of genetically modified foodGMF has become one of hot issue of paying close attention in the whole world; In order to identify genetically modified crops; Usually not only to screen and detect CaMV35S promotor and the NOS terminator of genetically modified crops, also will identify the strain and quantitative transgene component of transgenic crop.
The research of deep processed product PCR molecular detection technology is the difficult point and the focus of detection technique research always; Because its genomic DNA fragment is because the destruction of course of processing mesohigh high temperature and mechanical force; Be degraded into very small segment (＜100bp); And content is very small, so traditional P CR-agarose gel electrophoresis can't false-negative result occur through regular meeting to these little purpose fragment realization detections effectively.
At present, the existing multi-PRC reaction technology that adopts increases to these small segment genes, to improve detection specificity.But because the multi-PRC reaction system is comparatively complicated, follow-up separation method is required strictness more, traditional agarose gel electrophoresis carries out compartment analysis, and its resolving power is limited, can't reach the requirement that lower molecular multiplex PCR-amplified fragments is detected.
Therefore, need a kind of ability of research quickness and high efficiency ground carry out isolating method to lower molecular multiplex PCR-amplified fragments.
Summary of the invention
The objective of the invention is to overcome the defective of the method for existing detection PCR small molecules amplified fragments, a kind of method of easy and simple to handle, separation lower molecular multiplex PCR-amplified fragments rapidly and efficiently is provided.
The efficient separation method that is used for lower molecular multiplex PCR-amplified fragments provided by the invention, it carries out purifying with the magnetic beads for purifying method to lower molecular multiplex PCR-amplified fragments earlier, and then adopts capillary electrophoresis to separate.
Sample according to the invention is any raw produce and deep processed product that contains the corn composition in the field of food.
Because the multi-PRC reaction system is comparatively complicated, has the impurity that some may interfere with follow-up capillary electrophoresis, therefore need carry out purification process to it.The present invention adopts the magnetic beads for purifying method.Magnetic bead is a kind of functional supports that is coated with bio-active group, can be scattered in to form the magnetic liquid material in the base fluid, and it has the flowability of liquid and the dual characteristics of solid magnetic particulate material concurrently, thereby makes the separation of solid-liquid phase become very convenient quick.The magnetic beads for purifying method is organically integrated DNA stripping technique and beneficiation technologies specifically, promptly can obtain the very high target matter DNA of purity through simple wash-out.
Capillary electrophoresis (Capillary Electrophoresis; CE) also be HPCE (HPCE); Capillary electrophoresis is development in recent years one of separate analytical technique rapidly; Capillary electrophoresis has overcome that traditional agarose gel electrophoresis resolving power is lower, process is complicated, the sample requirement is big, poor reproducibility, and its topmost characteristics are efficient, quick and micro-.That the present invention adopts is non gel sieving capillary gel electrophoresis (NGE), and its adopts is can form gel-free but non gel sieving medium that sieving action is arranged, and it can avoid the formation of cavity, and is simpler, convenient than the preparation of gel column.As far back as nineteen ninety, existing SEPIGEL 305 (LPA) is as the report of DNA separating medium.But its viscosity is big, operates more loaded down with trivial detailsly again, and is not very stable.So at several years thereafter, PEO, PDMA, PVP were in the news at the linear polymeric material that the fused quartz capillary wall has dynamic coating effect separately successively.Through experimental study, it is Natvosol (HEC) that the present invention selects separating medium.
The magnetic bead that said magnetic beads for purifying method adopts BIO-V company to buy carries out purifying.Adopt magnetic bead absorption, washing and elution step successively.
The step of said magnetic beads for purifying method is:
1) gets multiple PCR products and add magnetic bead combination liquid (to 100mmol/L Tris-Cl; Add 2.0mol/L NaCl, 1.0mol/L MgCl2,15% PEG 6000 and 15% PEG 8000 in the 10mmol/L EDTA solution, the pH value is 6.5) and the DTT solution (WR 34678 solution) of 30uL 1mol/L;
2) to wherein adding the magnetic bead that keeps even suspended state, make magnetic bead keep suspended state 5min;
3) centrifuge tube is placed carry out magnetic resolution on the magnet stand, inhale and remove liquid;
4) add 70% ethanol then and wash mixing; Carry out magnetic resolution, inhale and remove liquid;
5) repeating step 4) once;
6) under the magnetic condition, room temperature is dried, and inhales and remove liquid;
7) add TE (pH 8.0 for 100mmol/L Tris-Cl, 10mmol/L EDTA) solution, mixing;
8) magnetic resolution, imbitition, gained liquid are the PCR product of purifying.
The concrete operations step is following:
1) gets multiple PCR products in 1.5ml Eppendorf centrifuge tube.Adding isopyknic magnetic bead combines liquid (to 100mmol/L Tris-Cl, to add 2.0mol/L NaCl, 1.0mol/L MgCl in the 10mmol/L EDTA solution
2, 15% PEG 6000 and 15% PEG 8000, the pH value is 6.5) and the DTT solution of 30uL 1mol/L.
2) add the 200uL magnetic bead.Before drawing magnetic bead, the reagent bottle 30s of necessary first vortex vibration dress magnetic bead, even to guarantee the magnetic bead suspended state.Blow and beat repeatedly or, make magnetic bead remain suspended state 5min with liquid getting device with the abundant mixing of whirlpool mixed instrument.
3) centrifuge tube is positioned over leaves standstill 30s on the magnet stand, treat that magnetic bead is adsorbed in when centrifuge tube leans on magnet stand one side fully and can inhale liquid, liquid feed is drawn clean.
4) take off centrifuge tube from magnet stand, the ethanol that adds 1mL 70% in the centrifuge tube washs, concussion mixing 1min.Centrifuge tube is positioned on the magnet stand, and magnetic resolution 30s inhales as far as possible and removes liquid.
5) repeating step 4) once.
6) centrifuge tube is placed on the magnet stand all the time, and room temperature was dried 10～15 minutes, and is every at a distance from 5min, may be from magnetic bead liquid body exudate, accumulate in the centrifuge tube bottom, with liquid getting device these liquid are in time taken out.
7) (pH8.0) solution shakes mixing 5～10min, makes magnetic bead remain suspended state for 100mmol/L Tris-Cl, 10mmol/L EDTA to add TE to centrifuge tube.
8) magnetic separates, and centrifuge tube is positioned over leaves standstill about 30s on the magnet stand, and careful imbitition is put into new centrifuge tube when making magnetic bead be adsorbed in centrifuge tube by magnet stand one side fully, and gained liquid is the PCR product of purifying.
Natvosol (HEC) is a main research object of the present invention.The present invention with the linear polymeric solution of HEC as the filling medium in the kapillary.Utilization kapillary non gel sieving and capillary dynamic are coated with stain zone electrophoresis pattern and set up the scheme to the multiple PCR products screening.
The used screening solution system of capillary electrophoresis separation method of the present invention is made up of sieving medium and matrix damping fluid.Sieving medium is Natvosol (HEC), and the matrix damping fluid is made up of 89mol/L Tris base (Tutofusin tris), 332mol/L boric acid and 2mol/L EDTA (YD 30), and pH further is preferably 7.4 between 7.2～7.5.Wherein, Tris, boric acid and EDTA are the analytical pure rank at least.
Described sieving medium HEC is a water-soluble high-molecular substance, and preferred mass concentration scope further is preferably 1.2% 1.0～1.5%.Capillary inner diameter is 75 μ m.Confirm that through optimizing the back use length to be 48.5cm, the distance of detection window to electrode end is 8.5cm, so the kapillary useful length is 40cm at every turn.Running voltage is a negative pressure, and size further is optimized for-16.5kv between 15kv～20kv.
Specifically, Capillary Electrophoresis Separation Process according to the invention is:
1) preparation electrophoretic buffer: with 30mL solution is example, takes by weighing the Tris of 323.4mg, the boric acid of 615.9mg, and the EDTA of 22.5mg is settled to 30mL with deionized water, regulates pH value to 7.2～7.5 with the boric acid of 1.0mol/L.
2) with 1) in the electrophoretic buffer branch take on 15mL and prepare dissociating buffer.To wherein adding mass concentration is 1.0～1.5% HEC because the viscosity of HEC is bigger, so be difficult for dissolving, can adopt the concussion of whirlpool oscillator after, put into water-bath temperature again and bathe and make it to be dissolved in fully electrophoretic buffer, be cooled to 4 ℃ and seal preservation.
3) with 1) and 2) solution cross 45 μ m filter membranes, 4 ℃ of preservations.
4) internal diameter capillaceous is 75 μ m, and at 8.2～8.5cm place calcination kapillary, as detection window, capillary pipe length is 48.5cm, and useful length is 40cm.
5) calcination head and tail capillaceous place makes it pass electrode and immerses in the electrophoretic buffer.
6) condition of new pre-treatment capillaceous is following: the sodium hydroxide operation 5min of 0.10mol/L; Deionized water operation 10min; Dissociating buffer operation 10min.
7) parameter of HPCE operation is following: capillary temperature is 20 ℃, sample introduction 20s under the-5kv, and the operation down of the voltage of-16.5kv detects under Sig.260/8nm and Ref.350/80nm condition.
The efficient separation method that is used for lower molecular multiplex PCR-amplified fragments of the present invention can extract Plant Genome earlier from sample; Carry out the multiplex PCR amplification then.
The present invention extracts plant genome DNA and adopts CTAB (bromohexadecane base Trimethylamine 99) method from sample.Concrete steps are following:
The crispy rice sample that 1) will contain the corn composition is ground into powder in liquid nitrogen.
2) taking by weighing 200mg joins in the 1.5mL Eppendof centrifuge tube (safe Eppendorf tube).
3) add 1000 μ LCTAB solution, vibration evenly, 65 ℃ of incubation 40min, mixing 5 times at least therebetween was centrifugal 10 minutes of 4 ℃ of following 12000rpm.
4) carefully draw supernatant in another new pipe, add and the saturated phenol of the isopyknic Tris of supernatant: trichloromethane: primary isoamyl alcohol (25: 24: 1), fully vibration is even, 4 ℃ of centrifugal 15min of following 12000rpm.
5) repeating step 4) once.
6) carefully draw supernatant in another new pipe, add and the isopyknic trichloromethane of supernatant: primary isoamyl alcohol (24: 1), vibration is even, at 4 ℃ of centrifugal 15min of following 12000rpm.
7) carefully draw supernatant in another new pipe, add the Virahol of 2/3 volume, the sodium acetate of 1/10 volume.Leave standstill 2h under-20 ℃ of conditions.At 4 ℃ of centrifugal 15min of following 12000rpm.
8) abandoning supernatant adds 70% an amount of ethanolic soln washing precipitation.
9) abandoning supernatant dries up deposition.With the aseptic ultrapure water dissolution precipitation of 100uL.
10) add 3 μ l RNA enzyme solution, digest 30min in 37 ℃ of water-bath environment.
Sample of the present invention can be crispy rice, also can be any deep processed product and deep processing transgenic product that contains the corn composition in the field of food.
The segmental amplification procedure of lower molecular multiplex PCR of the present invention is:
The PCR reaction system is (30 μ L) as follows: 10 * PCR damping fluid: 3 μ L (10 * PCR damping fluid: 100mmol/L Tris-HCl, 500mmol/L KCl, 15mmol/L MgCl
2); 2.5mmol/L dNTP:2.4 μ L; Primer concentration is 10 μ M, totally three pairs of primers, and the upstream and downstream primer of every pair of primer adds 1 μ L respectively; The Taq archaeal dna polymerase: 0.4 μ L, the DNA of extraction add 1 μ l as template, use ddH
2O water complements to 30 μ l.
The amplification condition of PCR is following: sex change in advance: 94 ℃ of 5min; Sex change: 94 ℃ of 30s; Annealing: 60 ℃ of 30s; Extend: 72 ℃ of 30s; Extend at last: 72 ℃ of 7min; Establish 35 circulations altogether.
The present invention adopts the paramagnetic particle method purified pcr product; Efficiently removed the impurity in the reaction system fast; Make it be suitable for carrying out capillary electrophoresis, appearance is gone up in the PCR product dilution back that will pass through behind the purifying, sharp peak occurred; It is thus clear that magnetic beads for purifying helps the operation of capillary electrophoresis, improved the purity of sample greatly.
The magnetic beads for purifying method that adopts among the present invention is different from common DNA test kit method of purification; The principle that common method of purification utilizes the silicon class matrix under the high salt concentration that the DNA uniqueness is adsorbed; Remove gel and other impurity, lower to the recovery less than the dna fragmentation of 100bp.And the magnetic beads for purifying method of using among the present invention is foundation with the unique centrifugation of magnetic bead, an extremely easy schedule of operation is provided: magnetic bead absorption, washing and wash-out.Under the righttest reagent and operational condition, can obtain high-quality DNA.The present invention adopts the magnetic beads for purifying method to have easy, quick and characteristics of high efficiency, need not use organic solvents such as phenol, chloroform in the whole extraction purge process.
The invention provides the capillary electrophoresis that is used to separate small segment PCR product, this method can be in 10.6min under the ultraviolet detection condition success isolate the lower molecular multiplex PCR-amplified fragments that size is respectively 51bp, 74bp and 88bp.
The implementation result of table 1 the present invention and traditional agarose gel electrophoresis relatively
The present invention has remedied the deficiency that prior art detects lower molecular multiplex PCR-amplified fragments, successful foundation a kind of quick screening method that is used for deep processed product PCR Molecular Detection.Multiple PCR technique has the template of saving, saves reagent and the high efficiency of time; Paramagnetic particle method is handled multiple PCR products can effectively remove the impurity in the PCR reaction system, and it is more suitable in carrying out capillary electrophoresis; Capillary electrophoresis has the advantages that detectability is low, highly sensitive and detection time is short.Application safety of the present invention, can success lower molecular multiplex PCR-amplified fragments is carried out isolation identification, be controlled at 10.6min detection time, can be widely used in the fields such as food sanitation quarantine and Clinical Laboratory.
Description of drawings
Fig. 1 is agarose gel electrophoresis checking extracting genome DNA figure of the present invention; M: λ DNA/EcoR I+Hind III Marker, 1,2 are the crispy rice food DNA electrophoretogram that the CTAB method is extracted
Fig. 2 detects multiple PCR products for agarose gel electrophoresis of the present invention; M:DL 2000DNA Marker, 1,2 are multiple PCR products
Fig. 3 is a capillary electrophoresis separation multiple PCR products of the present invention;
Fig. 4 goes out the enlarged view of peak part for Fig. 3.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Embodiment 1 usefulness CTAB method is extracted genomic dna from crispy rice
The crispy rice sample that 1) will contain the corn composition is ground into powder in liquid nitrogen.
2) take by weighing 200mg and join in the 1.5mL Eppendof centrifuge tube, need set up negative control simultaneously.
3) add 1000 μ L CTAB solution, vibration evenly, 65 ℃ of incubation 40min, mixing 5 times at least therebetween was centrifugal 10 minutes of 4 ℃ of following 12000rpm.
4) carefully draw supernatant in another new pipe, add and the saturated phenol of the isopyknic Tris of supernatant: trichloromethane: primary isoamyl alcohol (25: 24: 1), fully vibration is even, 4 ℃ of centrifugal 15min of following 12000rpm.
5) repeating step (4) once.
6) carefully draw supernatant in another new pipe, add and the isopyknic trichloromethane of supernatant: primary isoamyl alcohol (24: 1), vibration is even, at 4 ℃ of centrifugal 15min of following 12000rpm.
7) carefully draw supernatant in another new pipe, add the Virahol of 2/3 volume, the sodium acetate of 1/10 volume.Leave standstill 2h under-20 ℃ of conditions.At 4 ℃ of centrifugal 15min of following 12000rpm.
8) abandoning supernatant adds 70% an amount of ethanolic soln washing precipitation.
9) abandoning supernatant dries up deposition.With the aseptic ultrapure water dissolution precipitation of 100uL.
10) add 3 μ l RNA enzyme solution, digest 30min in 37 ℃ of water-bath environment.
11) after the digestion, run the agarose gel electrophoresis of 1% concentration the genome that is extracted is verified.
As shown in Figure 1, the result shows, finds that adopt the CTAB method can extract the genomic dna in the crispy rice, applied sample amount is 5 μ L after during through agarose gel electrophoresis.The position of wherein genomic size about the 21kb of λ DNA/EcoR I+Hind III Marker.Genomic band disperse, this is the result who adopts after deep process technology is handled sample, does not have albumen residual basically in the sample point sample hole, extracts the genome that obtains with method of the present invention and all can be used for pcr amplification.
The DNA that 2 couples of embodiment of embodiment 1 extract is PCR and detects
The DNA that uses embodiment 1 to extract is template, with the genome of designed primer (like table 2) amplification crispy rice.The PCR reaction system is (30 μ L) as follows: 10 * PCR damping fluid: 3 μ L (10 * PCR damping fluid: 100mmol/L Tris-HCl, 500mmol/L KCl, 15mmol/L MgCl
2); 2.5mmol/L dNTP:2.4 μ L; Primer concentration is 10 μ M, totally three pairs of primers, and the upstream and downstream primer of every pair of primer adds 1 μ L respectively; The Taq archaeal dna polymerase: 0.4 μ L, the DNA of extraction add 1 μ l as template, use ddH
2O water complements to 30 μ l.The amplification condition of PCR is following: sex change in advance: 94 ℃ of 5min; Sex change: 94 ℃ of 30s; Annealing: 60 ℃ of 30s; Extend: 72 ℃ of 30s; Extend at last: 72 ℃ of 7min; Establish 35 circulations altogether.
Table 2 design of primers
As shown in Figure 2, agarose gel electrophoresis detects multiple PCR products, and applied sample amount is 8 μ L.The result shows; Detect multiple PCR products with agarose gel electrophoresis; The target DNA fragment size of multiplex PCR amplification is respectively 51bp, 74bp and 88bp; Product accumulates in the part below the 100bp of DL 2000DNAMarker with the bulk form, visible traditional agarose gel electrophoresis can't effectively separate it.Wherein DL 2000DNA Marker is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom.
Embodiment 3 adopts the magnetic beads for purifying method that lower molecular multiplex PCR-amplified fragments is carried out purifying
1) gets multiple PCR products in 1.5ml Eppendorf centrifuge tube.Adding isopyknic magnetic bead combines liquid (to 100mmol/L Tris-Cl; Add 2.0mol/L NaCl, 1.0mol/L MgCl2,15% PEG 6000 and 15% PEG 8000 in the 10mmol/L EDTA solution, the pH value is 6.5) and the DTT solution of 30uL 1mol/L;
2) add the 200uL magnetic bead.Before drawing magnetic bead, the reagent bottle 30s of necessary first vortex vibration dress magnetic bead, even to guarantee the magnetic bead suspended state.Blow and beat repeatedly or, make magnetic bead remain suspended state 5min with liquid getting device with the abundant mixing of whirlpool mixed instrument.
3) centrifuge tube is positioned over leaves standstill 30s on the magnet stand, treat that magnetic bead is adsorbed in when centrifuge tube leans on magnet stand one side fully and can inhale liquid, liquid feed is drawn clean.
4) take off centrifuge tube from magnet stand, the ethanol that adds 1mL 70% in the centrifuge tube washs, concussion mixing 1min.Centrifuge tube is positioned on the magnet stand, and magnetic resolution 30s inhales as far as possible and removes liquid.
5) repeating step 4) once.
6) centrifuge tube is placed on the magnet stand all the time, and room temperature was dried 10～15 minutes, and is every at a distance from 5min, may be from magnetic bead liquid body exudate, accumulate in the centrifuge tube bottom, with liquid getting device these liquid are in time taken out.
7) (pH8.0) solution shakes mixing 5～10min, makes magnetic bead remain suspended state for 100mmol/L Tris-Cl, 10mmol/L EDTA to add TE to centrifuge tube.
8) magnetic separates, and centrifuge tube is positioned over leaves standstill about 30s on the magnet stand, and careful imbitition is put into new centrifuge tube when making magnetic bead be adsorbed in centrifuge tube by magnet stand one side fully, and gained liquid is the PCR product of purifying.
1) preparation electrophoretic buffer: with 30mL solution is example, takes by weighing the Tris of 323.4mg, the boric acid of 615.9mg, and the EDTA of 22.5mg is settled to 30mL with deionized water, regulates pH value to 7.4 with the boric acid of 1.0mol/L.
2) with 1) in the electrophoretic buffer branch take on 15mL and prepare dissociating buffer.To the HEC that wherein adds 180.0mg.Because the viscosity of HEC is bigger, thus be difficult for dissolving, can adopt the concussion of whirlpool oscillator after, put into water-bath temperature again and bathe and make it to be dissolved in fully electrophoretic buffer, be cooled to 4 ℃ and seal preservation.
3) with 1) and 2) solution cross 45 μ m filter membranes, 4 ℃ of preservations.
4) internal diameter capillaceous is 75 μ m, and inside pipe wall does not have coating, and at 8.2～8.5cm place calcination kapillary, as detection window, capillary pipe length is 48.5cm, and useful length is 40cm.
5) calcination head and tail capillaceous place makes it pass electrode and immerses in the electrophoretic buffer.
6) condition of new pre-treatment capillaceous is following: the sodium hydroxide operation 5min of 0.10mol/L; Deionized water operation 10min; Dissociating buffer operation 10min.
7) parameter of HPCE operation is following: capillary temperature is 20 ℃, sample introduction 20s under the-5kv, and the operation down of the voltage of-16.5kv detects under Sig.260/8nm and Ref.350/80nm condition.
As shown in Figure 3, the result shows, after the dilution of the process of the PCR product behind the magnetic beads for purifying; Sample introduction 20s under the condition of-5kv, capillary electrophoresis can successfully isolate the PCR products of 3 different sizes below the 100bp, and the target DNA fragment size is respectively 51bp, 74bp and 88bp; Capillary electrophoresis is good to its isolating separating size; The purified back of sample sample introduction, peak shape is sharp peak, in 10.6min, reaches separation; It is thus clear that the purity of sample height very, magnetic beads for purifying method help to carry out separation capillaceous.
The present invention is used for the multi-PRC reaction system of transgenic plant examination; With transgenic corns Bt176 is research object; Adopt multiple PCR technique, a PCR reacts CaMV 35S promoter, NOS terminator and the ZSSIIb gene of the corn that increases simultaneously, to improve detection efficiency and detection specificity.And having adopted paramagnetic particle method that multiple PCR products is carried out purifying, this method is quick, effective, easy and simple to handle.A kind of kapillary glue-free sieving system that is used for the above-mentioned multiple PCR products of capillary electrophoresis separation is provided simultaneously, and the electrophoretic buffer of this sieving system is 89mol/L Tris, 332mol/L boric acid, and 2mol/L EDTA, PH are 7.4.Sieving medium is Natvosol (HEC).
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
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Claims (4)
1. an efficient separation method that is used for lower molecular multiplex PCR-amplified fragments is characterized in that, it carries out purifying with the magnetic beads for purifying method to lower molecular multiplex PCR-amplified fragments earlier, and then adopts capillary electrophoresis to separate; The purification step of said magnetic beads for purifying method is:
1) get multiple PCR products and add the DTT solution that magnetic bead combines liquid and 30uL 1mol/L, said magnetic bead combines being formulated as of liquid: to 100mmol/L Tris-Cl, add 2.0mol/L NaCl, 1.0mol/L MgCl in the 10mmol/L EDTA solution
2, 15% PEG 6000 and 15% PEG 8000, the pH value is 6.5;
2) to wherein adding the magnetic bead that keeps even suspended state, make magnetic bead keep suspended state 5min;
3) centrifuge tube is placed carry out magnetic resolution on the magnet stand, inhale and remove liquid;
4) add 70% ethanol then and wash mixing; Carry out magnetic resolution, inhale and remove liquid;
5) repeating step 4) once;
6) under the magnetic condition, room temperature is dried, and inhales and remove liquid;
7) add TE solution, mixing;
8) magnetic resolution, imbitition, gained liquid are the PCR product of purifying;
Said capillary electrophoresis process is:
1) preparation electrophoretic buffer: take by weighing the Tris of 323.4mg, the boric acid of 615.9mg, the EDTA of 22.5mg is settled to 30mL with deionized water, regulates pH value to 7.2～7.5 with the boric acid of 1.0mol/L;
2) with 1) in the electrophoretic buffer branch take on part and prepare dissociating buffer, be 1.0～1.5% HEC to wherein adding mass concentration, heating for dissolving is cooled to 4 ℃ of sealings afterwards and preserves;
3) with 1) and 2) solution cross 0.45 μ m filter membrane, 4 ℃ of preservations;
4) internal diameter capillaceous is 75 μ m, and at 8.2～8.5cm place calcination kapillary, as detection window, capillary pipe length is 48.5cm, and useful length is 40cm;
5) calcination head and tail capillaceous place makes it pass electrode and immerses in the electrophoretic buffer;
6) condition of new pre-treatment capillaceous is following: the sodium hydroxide operation 5min of 0.10mol/L; Deionized water operation 10min; Dissociating buffer operation 10min;
7) parameter of HPCE operation is following: capillary temperature is 20 ℃, sample introduction 20～40s under the-5kv ,-15～-the voltage operation down of 20kv, under Sig.260/8nm and Ref.350/80nm condition, detect.
2. separation method according to claim 1 is characterized in that, the pH value of electrophoretic buffer is 7.4.
3. separation method according to claim 1 and 2 is characterized in that, the mass concentration of HEC is 1.2%.
4. separation method according to claim 1 and 2 is characterized in that, sample injection time is 20s in the step 7), and voltage is-16.5kv.
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|---|---|---|---|---|
| CN105063015A (en) * | 2015-07-28 | 2015-11-18 | 福建师范大学 | Method for rapid purification of specific amplification PCR (polymerase chain reaction) product based on paramagnetic particle method |
| CN108949746A (en) * | 2018-07-23 | 2018-12-07 | 杭州和壹基因科技有限公司 | A kind of method of human faecal mass total DNA sulphite conversion and recovery purifying |
| CN111073886B (en) * | 2020-01-16 | 2023-08-29 | 中国农业科学院农业基因组研究所 | DNA extraction adsorption liquid based on superparamagnetism nano-particles, kit and DNA extraction method |
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2009
- 2009-11-05 CN CN2009102367300A patent/CN101705225B/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104419760A (en) * | 2013-09-07 | 2015-03-18 | 上海毕欧桥生物科技有限公司 | Sample purification method applied in analysis of nucleic acid sequence |
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