CN112941070A - Plasma free DNA extraction kit based on hydroxyl magnetic beads and use method - Google Patents
Plasma free DNA extraction kit based on hydroxyl magnetic beads and use method Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a plasma free DNA extraction kit based on hydroxyl magnetic beads, which comprises a kit, wherein the kit consists of a proteinase K solution, the hydroxyl magnetic beads, a lysis solution, a lysis binding solution, a primary washing solution, a secondary washing solution and an eluent, the magnetic beads in the kit are the hydroxyl magnetic beads with the particle size of 200-1000nm, and the concentration is 10-100 mg/ml; the lysis solution in the kit contains 100-200g/L sodium dodecyl sulfate, and the kit can be adapted to a high-flux automatic extraction instrument by designing a magnetic bead adsorption method, so that a better extraction effect can be achieved, the cost is low, the development trend of the current kit is better met, and meanwhile, a reagent combination scheme is designed according to the requirement of the magnetic bead adsorption method on the extraction effect, so that high extraction efficiency, high extraction lower limit and excellent stability are realized.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a plasma free DNA extraction kit based on hydroxyl magnetic beads and a using method thereof.
Background
In recent years, as the PCR detection technology is mature, the research on the genetic information expressed by human DNA is deepened in the medical community. Accordingly, more and more applications of predicting and diagnosing diseases through DNA are developed, wherein human plasma free DNA is an excellent research and clinical application sample due to low acquisition difficulty. Predicting diseases by reading the information expressed by plasma free DNA is also widely used clinically, for example: collecting peripheral blood of a pregnant woman, and carrying out identification analysis on free DNA of a fetus contained in the peripheral blood so as to predict noninvasive prenatal diagnosis of paternal genetic diseases of a newborn; collecting blood of a patient, and carrying out identification analysis on tumor DNA in free DNA so as to judge the disease condition of the patient.
To reach a reliable clinical conclusion, the extraction of free plasma DNA is an important step. The method for reliably extracting the free DNA of the blood plasma mainly comprises a silicon membrane centrifugal column adsorption method and a magnetic bead adsorption method, wherein the principle of the silicon membrane centrifugal column adsorption method is that nucleic acid is adsorbed to the surface of silicon dioxide, impurities such as chemicals, saccharides, proteins and the like are quickly washed away by using a washing solution, and then the nucleic acid is eluted by using an eluent. The main principle of the method is to introduce active groups on the surface of a magnetic bead (mainly ferrite) through copolymerization, surface modification and the like, wherein the active groups comprise hydroxyl, carboxyl, amino, sulfydryl and the like to prepare a magnetic affinity adsorbent, then after the magnetic bead is incubated with nucleic acid, the nucleic acid is adsorbed on the surface of the magnetic bead through principles of hydrophobic interaction, electrostatic interaction, ion adsorption and the like, and the magnetic bead compound with the nucleic acid bound on the surface is obtained through simple washing and magnetic separation, and the nucleic acid can be eluted from the magnetic bead by changing conditions such as pH value of a reagent and the like, but the method has the following defects:
in clinical application, the kit is selected mainly by considering the cost, the use cost and the extraction effect of the kit, wherein the extraction effect is particularly important, and the extraction effect is mainly evaluated by the extraction efficiency, the extraction amount and the extraction stability;
the silicon membrane centrifugal column adsorption method kit on the market has excellent extraction amount and extraction stability, but has obvious disadvantages in terms of kit cost and extraction efficiency compared with a magnetic bead method kit which can be used together with an instrument;
the magnetic bead adsorption method kit is matched with a high-flux automatic extraction instrument, so that a better extraction effect can be achieved, meanwhile, the cost is relatively low, the development trend of the current kit is met, and the development direction of a kit manufacturer is provided, but a certain difference exists between the manual extraction effect and a silicon membrane centrifugal column adsorption method.
In conclusion, the invention solves the existing problems by designing the plasma free DNA extraction kit based on the hydroxyl magnetic beads and the use method.
Disclosure of Invention
The invention aims to provide a plasma free DNA extraction kit based on hydroxyl magnetic beads and a using method thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a plasma free DNA extraction kit based on hydroxyl magnetic beads comprises a kit, wherein the kit consists of a proteinase K solution, the hydroxyl magnetic beads, a lysis solution, a lysis binding solution, a primary washing solution, a secondary washing solution and an eluent, wherein the magnetic beads in the kit are the hydroxyl magnetic beads with the particle size of 200-1000nm, and the concentration is 10-100 mg/ml;
the lysis solution in the kit contains 100-200g/L sodium dodecyl sulfate;
the lysis binding solution in the kit comprises 100-500g/L guanidinium isothiocyanate, 1-10g/L citric acid, 2-20g/L sodium citrate, 1-10g/L sodium chloride, 10-50% triton X-100 by volume and 10-50% isopropanol by volume;
the primary washing solution in the kit comprises 100-500g/L guanidinium isothiocyanate, 2-20g/L Tris, 2-20g/L EDTA, 10-50% by volume triton X-100 and 10-50% by volume ethanol;
the secondary washing solution in the kit contains 50-90% by volume of ethanol;
the eluent in the kit comprises 0.1-3g/L of disodium ethylene diamine tetraacetate and 0-5g/L of Tris.
Preferably, the proteinase K solution in the kit is 20mg/ml, the preparation is carried out before the kit is used, the effective period of the solution is 6 months at 4 ℃ and the solution is stored for a long time at-20 ℃, the particle size of magnetic beads in the kit is 400-1000nm, and the concentration is 20-60 mg/ml.
Preferably, the lysis solution in the kit contains 150-200g/L sodium dodecyl sulfate.
Preferably, the lysis binding solution in the kit comprises 300-500g/L guanidine isothiocyanate, 1-5g/L citric acid, 2-10g/L sodium citrate, 1-5g/L sodium chloride, 10-30% by volume triton X-100 and 10-30% by volume isopropanol.
Preferably, the primary washing solution in the kit comprises 200-400g/L guanidinium isothiocyanate, 10-20g/L Tris, 2-10g/L EDTA, 10-30% by volume triton X-100 and 30-50% by volume ethanol.
Preferably, the secondary washing solution in the kit comprises 70-90% ethanol by volume, and the eluent in the kit comprises 0.1-1.5g/L disodium edetate and 1-3g/L Tris
A method for using a plasma free DNA extraction kit based on hydroxyl magnetic beads takes 1ml plasma extraction as an example, the component solutions of other volumes of plasma are multiplied by corresponding times respectively, and the dosage of the first washing solution and the second washing solution is fixed to be 1ml except the first washing solution and the second washing solution, and the maximum dosage of the plasma is not more than 5 ml. Under the condition of extraction by using an instrument, adding a solution with a corresponding volume according to corresponding steps of manual extraction according to the instruction of the instrument, wherein the specific steps are as follows:
s1, cleavage:
(1) preparing 20mg/ml proteinase K solution before use, or taking out the prepared proteinase K solution to balance to room temperature. Taking the magnetic bead solution to balance to room temperature;
(2) adding 1ml of plasma into a 15ml centrifuge tube, adding 20ul of protease K solution, shaking for 5 seconds, adding 100ul of lysis solution, and shaking for 10 seconds;
(3) covering the centrifuge tube, and putting the centrifuge tube into a constant-temperature water bath kettle at 60 ℃ for incubation for 20 minutes;
s2, cleavage binding:
(4) mixing the magnetic beads with lysis binding solution, wherein the usage amount of the magnetic beads corresponding to 1ml of blood plasma and the lysis binding solution is 20ul and 1.25ml respectively;
(5) taking out the sample from the water bath after the incubation of the sample in the centrifuge tube is finished, cooling for 5 minutes, shaking up the mixed solution of the magnetic beads and the lysis binding solution, and adding the mixed solution into the centrifuge tube according to corresponding amount respectively;
(6) turning the centrifugal tube upside down to mix the liquid evenly, and shaking the centrifugal tube gently or rotating the centrifugal tube for 15 minutes or using a blood mixer;
(7) moving the centrifuge tube to a magnetic frame, carrying out magnetic attraction for 5 minutes, keeping the magnetic attraction state, pouring the liquid out of the centrifuge tube or sucking the liquid out of the centrifuge tube by using a pipette gun, and removing the liquid as clean as possible but ensuring that the magnetic beads cannot be removed;
s3, one wash:
(8) taking off the centrifuge tubes from the magnetic frame, adding 1ml of primary washing liquid into each centrifuge tube, shaking to suspend the magnetic beads in the primary washing liquid, and transferring the liquid into 1.5ml of centrifuge tubes;
(9) placing a 1.5ml centrifuge tube on a magnetic frame, magnetically sucking for 2 minutes, transferring clear liquid to the original 15ml centrifuge tube by using a pipette gun, shaking and cleaning a cover and residual magnetic beads on the tube wall, transferring liquid to the 1.5ml centrifuge tube on the magnetic frame again, and discarding the 15ml centrifuge tube;
(10) after magnetic attraction for 2 minutes, a clear liquid part is sucked out by using a liquid transfer gun;
(11) adding 1ml of primary washing liquid into the centrifugal tube again, shaking for 30 seconds, and centrifuging to enable liquid on the upper cover of the centrifugal tube to enter the tube;
(12) placing a 1.5ml centrifuge tube on a magnetic frame for magnetic absorption for 2 minutes, and sucking out a clear liquid part by using a pipette;
s4, secondary washing:
(13) adding 1ml of secondary washing liquid into a 1.5ml centrifuge tube, shaking for 30 seconds, and centrifuging;
(14) placing a 1.5ml centrifuge tube on a magnetic frame for magnetic absorption for 2 minutes, and sucking out a clear liquid part by using a pipette;
(15) repeating the steps (13) and (14);
s5, elution:
(16) centrifuging to collect the clear liquid at the bottom of the centrifuge tube, magnetically sucking for one minute, and sucking out the residual clear liquid with a pipette;
(17) keeping the magnetic attraction state, opening a cover of a 1.5ml centrifuge tube, and airing the magnetic beads for 5 minutes at room temperature;
(18) adding 20ul of eluent into a 1.5ml centrifuge tube, shaking the centrifuge tube for 5 minutes to fully mix the eluent with the magnetic beads, and eluting DNA adsorbed on the magnetic beads;
(19) centrifuging, magnetically attracting for 1 minute, and using a pipette to pipette clear liquid to obtain a final DNA sample.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the magnetic bead adsorption method kit can be matched with a high-flux automatic extraction instrument, so that a better extraction effect can be achieved, the cost is low, the development trend of the current kit is better met, and meanwhile, a reagent combination scheme is designed according to the requirement of the magnetic bead adsorption method kit on the extraction effect, so that high extraction efficiency, high extraction lower limit and excellent stability are realized.
Drawings
FIG. 1 is a PE Labchip GX Touch capillary electrophoresis qualitative map of formula A of the example of the present invention;
FIG. 2 is a PE Labchip GX Touch capillary electrophoresis qualitative map of formula B of the example of the present invention;
FIG. 3 is a PE Labchip GX Touch capillary electrophoresis qualitative map of formulation C of the present invention;
FIG. 4 is a PE Labchip GX Touch capillary electrophoresis qualitative map of formulation D of the present invention;
FIG. 5 is a PE Labchip GX Touch capillary electrophoresis qualitative map of formulation E of the present invention;
FIG. 6 is a PE Labchip GX Touch capillary electrophoresis qualitative map of formulation F of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the protection scope of the present invention.
Referring to fig. 1-6, the present invention provides a technical solution:
a plasma free DNA extraction kit based on hydroxyl magnetic beads comprises a kit, wherein the kit consists of a proteinase K solution, the hydroxyl magnetic beads, a lysis solution, a lysis binding solution, a primary washing solution, a secondary washing solution and an eluent, wherein the magnetic beads in the kit are the hydroxyl magnetic beads with the particle size of 200-1000nm, and the concentration is 10-100 mg/ml;
the lysis solution in the kit contains 100-200g/L sodium dodecyl sulfate;
the lysis binding solution in the kit comprises 100-500g/L guanidinium isothiocyanate, 1-10g/L citric acid, 2-20g/L sodium citrate, 1-10g/L sodium chloride, 10-50% triton X-100 by volume and 10-50% isopropanol by volume;
the primary washing solution in the kit comprises 100-500g/L guanidinium isothiocyanate, 2-20g/L Tris, 2-20g/L EDTA, 10-50% by volume triton X-100 and 10-50% by volume ethanol;
the secondary washing solution in the kit contains 50-90% by volume of ethanol;
the eluent in the kit comprises 0.1-3g/L of disodium ethylene diamine tetraacetate and 0-5g/L of Tris.
Furthermore, the proteinase K solution in the kit is 20mg/ml, the preparation is carried out before the kit is used, the effective period of the solution is 6 months at 4 ℃ and the solution is stored for a long time at-20 ℃, the particle size of magnetic beads in the kit is 400-1000nm, and the concentration is 20-60 mg/ml.
Further, the lysis solution in the kit contains 150-200g/L sodium dodecyl sulfate.
Further, the lysis binding solution in the kit comprises 300-500g/L guanidine isothiocyanate, 1-5g/L citric acid, 2-10g/L sodium citrate, 1-5g/L sodium chloride, 10-30% by volume triton X-100 and 10-30% by volume isopropanol.
Further, the primary washing solution in the kit comprises 200-400g/L guanidinium isothiocyanate, 10-20g/L Tris, 2-10g/L EDTA, 10-30% triton X-100 by volume and 30-50% by volume ethanol.
Further, the secondary washing solution in the kit comprises ethanol with the volume of 70-90%, and the eluent in the kit comprises 0.1-1.5g/L of disodium edetate and 1-3g/L of Tris
A method for using a plasma free DNA extraction kit based on hydroxyl magnetic beads takes 1ml plasma extraction as an example, the component solutions of other volumes of plasma are multiplied by corresponding times respectively, and the dosage of the first washing solution and the second washing solution is fixed to be 1ml except the first washing solution and the second washing solution, and the maximum dosage of the plasma is not more than 5 ml. Under the condition of extraction by using an instrument, adding a solution with a corresponding volume according to corresponding steps of manual extraction according to the instruction of the instrument, wherein the specific steps are as follows:
s1, cleavage:
(1) preparing 20mg/ml proteinase K solution before use, or taking out the prepared proteinase K solution to balance to room temperature. Taking the magnetic bead solution to balance to room temperature;
(2) adding 1ml of plasma into a 15ml centrifuge tube, adding 20ul of protease K solution, shaking for 5 seconds, adding 100ul of lysis solution, and shaking for 10 seconds;
(3) covering the centrifuge tube, and putting the centrifuge tube into a constant-temperature water bath kettle at 60 ℃ for incubation for 20 minutes;
s2, cleavage binding:
(4) mixing the magnetic beads with lysis binding solution, wherein the usage amount of the magnetic beads corresponding to 1ml of blood plasma and the lysis binding solution is 20ul and 1.25ml respectively;
(5) taking out the sample from the water bath after the incubation of the sample in the centrifuge tube is finished, cooling for 5 minutes, shaking up the mixed solution of the magnetic beads and the lysis binding solution, and adding the mixed solution into the centrifuge tube according to corresponding amount respectively;
(6) turning the centrifugal tube upside down to mix the liquid evenly, and shaking the centrifugal tube gently or rotating the centrifugal tube for 15 minutes or using a blood mixer;
(7) moving the centrifuge tube to a magnetic frame, carrying out magnetic attraction for 5 minutes, keeping the magnetic attraction state, pouring the liquid out of the centrifuge tube or sucking the liquid out of the centrifuge tube by using a pipette gun, and removing the liquid as clean as possible but ensuring that the magnetic beads cannot be removed;
s3, one wash:
(8) taking off the centrifuge tubes from the magnetic frame, adding 1ml of primary washing liquid into each centrifuge tube, shaking to suspend the magnetic beads in the primary washing liquid, and transferring the liquid into 1.5ml of centrifuge tubes;
(9) placing a 1.5ml centrifuge tube on a magnetic frame, magnetically sucking for 2 minutes, transferring clear liquid to the original 15ml centrifuge tube by using a pipette gun, shaking and cleaning a cover and residual magnetic beads on the tube wall, transferring liquid to the 1.5ml centrifuge tube on the magnetic frame again, and discarding the 15ml centrifuge tube;
(10) after magnetic attraction for 2 minutes, a clear liquid part is sucked out by using a liquid transfer gun;
(11) adding 1ml of primary washing liquid into the centrifugal tube again, shaking for 30 seconds, and centrifuging to enable liquid on the upper cover of the centrifugal tube to enter the tube;
(12) placing a 1.5ml centrifuge tube on a magnetic frame for magnetic absorption for 2 minutes, and sucking out a clear liquid part by using a pipette;
s4, secondary washing:
(13) adding 1ml of secondary washing liquid into a 1.5ml centrifuge tube, shaking for 30 seconds, and centrifuging;
(14) placing a 1.5ml centrifuge tube on a magnetic frame for magnetic absorption for 2 minutes, and sucking out a clear liquid part by using a pipette;
(15) repeating the steps (13) and (14);
s5, elution:
(16) centrifuging to collect the clear liquid at the bottom of the centrifuge tube, magnetically sucking for one minute, and sucking out the residual clear liquid with a pipette;
(17) keeping the magnetic attraction state, opening a cover of a 1.5ml centrifuge tube, and airing the magnetic beads for 5 minutes at room temperature;
(18) adding 20ul of eluent into a 1.5ml centrifuge tube, shaking the centrifuge tube for 5 minutes to fully mix the eluent with the magnetic beads, and eluting DNA adsorbed on the magnetic beads;
(19) centrifuging, magnetically attracting for 1 minute, and using a pipette to pipette clear liquid to obtain a final DNA sample.
The specific implementation case is as follows:
the kit was formulated as follows. Collecting 6 different samples, respectively marking A-F, respectively storing by using a cfDNA storage tube of Streck company, separating each sample to obtain 2ml of blood plasma, respectively taking 1ml of blood plasma, extracting free DNA by using the kit and a American-based biological free DNA extraction kit (Cat number 12917 PC-100), quantitatively testing and comparing the concentrations of the extracted free DNA by using a Qubit 4 fluorescence quantitative meter, and qualitatively testing by using a PE Labchip GX Touch capillary electrophoresis apparatus.
The formula of the embodiment is as follows:
200g/L lysis solution sodium dodecyl sulfate
413.56g/L guanidine isothiocyanate, 1.536g/L citric acid, 6.46g/L trisodium citrate, 2.34g/L sodium chloride 20% (v/v) triton X-10030% (v/v) isopropanol
Primary washing solution 354.48g/L guanidinium isothiocyanate 12.114g/L Tris 7.306g/L EDTA 20% (v/v) triton X-10050% (v/v) ethanol
Secondary washing liquid 80% ethanol
Eluent 0.34g/L disodium ethylene diamine tetraacetate 1.21g/L Tris
The quantitive data of the Qubit 4 fluorometer are given in the following table:
PE Labchip GX Touch capillary electrophoresis qualitative map as follows, FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5 and FIG. 6:
according to the data, the invention has higher extraction concentration in extraction to meet the requirement of sequencing experiments such as PCR.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. The utility model provides a free DNA extraction kit of plasma based on hydroxyl magnetic bead, includes the kit, the kit comprises proteinase K solution, hydroxyl magnetic bead, lysate, schizolysis bonding liquid, washing liquid, secondary washing liquid and eluant, its characterized in that: the magnetic beads in the kit are hydroxyl magnetic beads with the particle size of 200 and 1000nm, and the concentration is 10-100 mg/ml;
the lysis solution in the kit contains 100-200g/L sodium dodecyl sulfate;
the lysis binding solution in the kit comprises 100-500g/L guanidinium isothiocyanate, 1-10g/L citric acid, 2-20g/L sodium citrate, 1-10g/L sodium chloride, 10-50% triton X-100 by volume and 10-50% isopropanol by volume;
the primary washing solution in the kit comprises 100-500g/L guanidinium isothiocyanate, 2-20g/L Tris, 2-20g/L EDTA, 10-50% by volume triton X-100 and 10-50% by volume ethanol;
the secondary washing solution in the kit contains 50-90% by volume of ethanol;
the eluent in the kit comprises 0.1-3g/L of disodium ethylene diamine tetraacetate and 0-5g/L of Tris.
2. The kit for extracting free DNA from plasma based on hydroxyl magnetic beads as claimed in claim 1, wherein: the proteinase K solution in the kit is 20mg/ml, the kit is prepared before use, the solution is stored for a long time at 4 ℃ and the validity period is 6 months and at-20 ℃, the particle size of magnetic beads in the kit is 400-1000nm, and the concentration is 20-60 mg/ml.
3. The kit for extracting free DNA from plasma based on hydroxyl magnetic beads as claimed in claim 1, wherein: the lysis solution in the kit contains 150-200g/L sodium dodecyl sulfate.
4. The kit for extracting free DNA from plasma based on hydroxyl magnetic beads as claimed in claim 1, wherein: the lysis binding solution in the kit comprises 300-500g/L guanidinium isothiocyanate, 1-5g/L citric acid, 2-10g/L sodium citrate, 1-5g/L sodium chloride, 10-30% triton X-100 by volume and 10-30% isopropanol by volume.
5. The kit for extracting free DNA from plasma based on hydroxyl magnetic beads as claimed in claim 1, wherein: the primary washing solution in the kit comprises 200-400g/L guanidinium isothiocyanate, 10-20g/L Tris, 2-10g/L EDTA, 10-30% by volume triton X-100 and 30-50% by volume ethanol.
6. The kit for extracting free DNA from plasma based on hydroxyl magnetic beads as claimed in claim 1, wherein: the secondary washing solution in the kit comprises ethanol with the volume of 70-90%, and the eluent in the kit comprises 0.1-1.5g/L of disodium edetate and 1-3g/L of Tris.
7. A use method of a plasma free DNA extraction kit based on hydroxyl magnetic beads takes 1ml plasma extraction as an example, other volumes of plasma are respectively multiplied by corresponding times, a first washing solution and a second washing solution are excluded, the use amount of the first washing solution and the second washing solution is fixed to be 1ml each time, the plasma is not more than 5ml at most, under the condition of extraction by using an instrument, the corresponding volumes of the solutions are added according to corresponding steps of manual extraction according to the instruction of the instrument, and the specific steps are as follows:
s1, cleavage:
(1) preparing 20mg/ml proteinase K solution before use, or taking out the prepared proteinase K solution to balance to room temperature, and taking out the magnetic bead solution to balance to room temperature;
(2) adding 1ml of plasma into a 15ml centrifuge tube, adding 20ul of protease K solution, shaking for 5 seconds, adding 100ul of lysis solution, and shaking for 10 seconds;
(3) covering the centrifuge tube, and putting the centrifuge tube into a constant-temperature water bath kettle at 60 ℃ for incubation for 20 minutes;
s2, cleavage binding:
(4) mixing the magnetic beads with lysis binding solution, wherein the usage amount of the magnetic beads corresponding to 1ml of blood plasma and the lysis binding solution is 20ul and 1.25ml respectively;
(5) taking out the sample from the water bath after the incubation of the sample in the centrifuge tube is finished, cooling for 5 minutes, shaking up the mixed solution of the magnetic beads and the lysis binding solution, and adding the mixed solution into the centrifuge tube according to corresponding amount respectively;
(6) turning the centrifugal tube upside down to mix the liquid evenly, and shaking the centrifugal tube gently or rotating the centrifugal tube for 15 minutes or using a blood mixer;
(7) moving the centrifuge tube to a magnetic frame, carrying out magnetic attraction for 5 minutes, keeping the magnetic attraction state, pouring the liquid out of the centrifuge tube or sucking the liquid out of the centrifuge tube by using a pipette gun, and removing the liquid as clean as possible but ensuring that the magnetic beads cannot be removed;
s3, one wash:
(8) taking off the centrifuge tubes from the magnetic frame, adding 1ml of primary washing liquid into each centrifuge tube, shaking to suspend the magnetic beads in the primary washing liquid, and transferring the liquid into 1.5ml of centrifuge tubes;
(9) placing a 1.5ml centrifuge tube on a magnetic frame, magnetically sucking for 2 minutes, transferring clear liquid to the original 15ml centrifuge tube by using a pipette gun, shaking and cleaning a cover and residual magnetic beads on the tube wall, transferring liquid to the 1.5ml centrifuge tube on the magnetic frame again, and discarding the 15ml centrifuge tube;
(10) after magnetic attraction for 2 minutes, a clear liquid part is sucked out by using a liquid transfer gun;
(11) adding 1ml of primary washing liquid into the centrifugal tube again, shaking for 30 seconds, and centrifuging to enable liquid on the upper cover of the centrifugal tube to enter the tube;
(12) placing a 1.5ml centrifuge tube on a magnetic frame for magnetic absorption for 2 minutes, and sucking out a clear liquid part by using a pipette;
s4, secondary washing:
(13) adding 1ml of secondary washing liquid into a 1.5ml centrifuge tube, shaking for 30 seconds, and centrifuging;
(14) placing a 1.5ml centrifuge tube on a magnetic frame for magnetic absorption for 2 minutes, and sucking out a clear liquid part by using a pipette;
(15) repeating the steps (13) and (14);
s5, elution:
(16) centrifuging to collect the clear liquid at the bottom of the centrifuge tube, magnetically sucking for one minute, and sucking out the residual clear liquid with a pipette;
(17) keeping the magnetic attraction state, opening a cover of a 1.5ml centrifuge tube, and airing the magnetic beads for 5 minutes at room temperature;
(18) adding 20ul of eluent into a 1.5ml centrifuge tube, shaking the centrifuge tube for 5 minutes to fully mix the eluent with the magnetic beads, and eluting DNA adsorbed on the magnetic beads;
(19) centrifuging, magnetically attracting for 1 minute, and using a pipette to pipette clear liquid to obtain a final DNA sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114231526A (en) * | 2022-02-23 | 2022-03-25 | 南京瑞贝西生物科技有限公司 | Method for extracting genome DNA of high-abundance fecal microorganisms |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114231526A (en) * | 2022-02-23 | 2022-03-25 | 南京瑞贝西生物科技有限公司 | Method for extracting genome DNA of high-abundance fecal microorganisms |
| CN114231526B (en) * | 2022-02-23 | 2022-08-16 | 南京瑞贝西生物科技有限公司 | Method for extracting genome DNA of high-abundance fecal microorganisms |
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