CN107245486A - Poba gene group DNA extraction method - Google Patents
Poba gene group DNA extraction method Download PDFInfo
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- CN107245486A CN107245486A CN201710473677.0A CN201710473677A CN107245486A CN 107245486 A CN107245486 A CN 107245486A CN 201710473677 A CN201710473677 A CN 201710473677A CN 107245486 A CN107245486 A CN 107245486A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
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Abstract
The present invention provides a kind of poba gene group DNA extraction method.The poba gene group DNA extraction method includes extracting blood sample, added to the step such as premixed liquid, heating reverse mixing, mixed liquor brief centrifugation.The DNA extraction method that the poba gene group DNA extraction method that the present invention is provided solves prior art wastes time and energy, the big technical problem of consumptive material consumption.
Description
Technical field
Field is extracted the present invention relates to DNA, and in particular to a kind of poba gene group DNA extraction method.
Background technology
In current poba gene test experience, common method is that purchase nucleic acid extraction kit is extracted or purified.
But whole extraction process will by lysate cracking → incubation → centrifugation → Organic extraction (or crossing post) → rinsing for several times →
Flow as drying → dissolving (or elution), finally gives DNA.Whole extraction process complex steps, consumptive material consumption is big,
Stand-by period is very long.
The content of the invention
DNA extraction method for solution prior art is wasted time and energy, and the big technical problem of consumptive material consumption, the present invention is provided
A kind of poba gene group DNA extraction method solved the above problems.
A kind of poba gene group DNA extraction method, poba gene need to be used with the poba gene group DNA extraction method
Group DNA extraction kit, the poba gene group DNA extraction kit includes following components：
Buffer solution GB, buffer B D, buffer solution GDB, rinsing liquid PWB, elution buffer TB, Proteinase K, adsorption column, receipts
Collector, centrifuge tube；
The poba gene group DNA extraction method comprises the following steps：
Step 1：Extract 200 μ L blood sample；
Step 2：Proteinase K described in buffer solution GB described in 200 μ L and 20 μ L is combined as premixed liquid, by the blood sample
Add into the premixed liquid, fully vibration is mixed, and obtains the first mixed liquor；
Step 3：First mixed liquor at 56 DEG C in placing certain time, while reverse mix, until described first mixes
Close liquid and be changed into clear liquid；
Step 4：First mixed liquor is placed into 2-5min at room temperature, buffer B D described in 350 μ L is added, fully
Brief centrifugation after reverse mixing, obtains the second mixed liquor；
Step 5：Second mixed liquor is added into the adsorption column, and the adsorption column is inserted in the collecting pipe, in
30sec is centrifuged under 12000rpm, the adsorption column is taken out, outwells the waste liquid in the collecting pipe, and the adsorption column is put back to
The collecting pipe；
Step 6：Buffer solution GDB described in 500 μ L is added into the adsorption column, in centrifuging 30sec under 12000rpm, is taken
Go out the adsorption column, outwell the waste liquid in the collecting pipe, and the adsorption column is put back into the collecting pipe；
Step 7：Rinsing liquid PWB described in 600 μ L is added into the adsorption column, in centrifuging 30sec under 12000rpm, is taken
Go out the adsorption column, outwell the waste liquid in the collecting pipe, and the adsorption column is put back into the collecting pipe；
Step 8：Rinsing liquid PWB described in 550 μ L is added into the adsorption column, in centrifuging 2.5min under 12000rpm, is taken
Go out the adsorption column, outwell the waste liquid in the collecting pipe, the adsorption column is inserted in the centrifuge tube；
Step 9：The lid of the adsorption column is opened, 2min is placed at room temperature；
Step 10：Elution buffer TB described in 50-200 μ L is vacantly added dropwise in the adsorbed film centre position of the adsorption column,
Room temperature is placed after 2min, and in centrifuging 2min under 12000rpm, detection sample is obtained in the centrifuge tube.
In a kind of preferred embodiment for the poba gene group DNA extraction method that the present invention is provided, the poba gene group
DNA extraction kit also includes buffer solution GS；
In the step one, when the amount of blood sample is less than 200 μ L, the buffer solution GS to 200 μ L is supplemented.
In a kind of preferred embodiment for the poba gene group DNA extraction method that the present invention is provided, the poba gene group
DNA extraction kit also includes cell pyrolysis liquid CL；
In the step one, when the amount of blood sample, which is more than 200 μ L, is less than 1mL, the step one is specifically divided into following
Step：
Step 1.1：The cell pyrolysis liquid of the 1-2.5 times of blood sample volume is added in the blood sample
CL, overturns after mixing, in centrifuging 1min under 10000rpm, sucks supernatant liquor, leaves nucleus precipitation；
Step 1.2：Buffer solution GS described in 200 μ L is added in nucleus precipitation.
In a kind of preferred embodiment for the poba gene group DNA extraction method that the present invention is provided, described in the step 4
A certain amount of flocculent deposit is included in second mixed liquor；
To in the lump it be added to the adsorption column comprising second mixed liquor including the flocculent deposit in the step 5
In.
Compared to prior art, the poba gene group DNA extraction method that the present invention is provided has the advantages that：
First, it is described using the blood sample is instilled in the step 2 of the poba gene group DNA extraction method
Mode in premixed liquid, solves the problem of often plus once the Proteinase K just needs to change a pipette tips, effectively reduces rifle
The consumption of head, alleviates the labor intensity of operating personnel.
2nd, in the step 4 of the poba gene group DNA extraction method, the buffer B D and first mixing
After the reverse mixing of liquid, also carrying out a brief centrifugation makes the two fully combine, and accelerates mixing time, prevents what is remained by lid
Liquid and cause the cross pollution between sample, and in the step 5 and subsequent step can obtain preferably separation or rinse imitate
Really.
3rd, in the step 8 of the poba gene group DNA extraction method, the use of the rinsing liquid PWB is suitably reduced
Amount, and extend centrifugation time, rinsing and centrifugally operated that originally need to repeatedly is reduced to the step 7 and the step 8
Two steps, and the purity of the detection sample is not influenceed；
Due to the reduction of the rinsing liquid PWB consumptions, the rinsing liquid will not be infected with again when taking out the adsorption column
PWB, without the operation individually once centrifuged again, simplifies operating procedure, shortens the extraction operating time, and to a certain degree
Improve the detection sample purity.
Embodiment
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of embodiment of the present invention, rather than whole embodiments.
Include with poba gene group DNA extraction kit used in the poba gene group DNA extraction method following
Component：
Cell pyrolysis liquid CL, buffer solution GS, buffer solution GB, buffer B D, buffer solution GDB, rinsing liquid PWB, elution buffer
Liquid TB, Proteinase K, adsorption column, collecting pipe, centrifuge tube.
The poba gene group DNA extraction method comprises the following steps：
Step 1：Extract 200 μ L blood sample；
Step 2：Proteinase K described in buffer solution GB described in 200 μ L and 20 μ L is combined as premixed liquid, by the blood sample
Add into the premixed liquid, fully vibration is mixed, and obtains the first mixed liquor；
Step 3：First mixed liquor at 56 DEG C in placing 10min, while reverse mix, first mixed liquor is changed into
Clear liquid；
Step 4：First mixed liquor is placed into 5min at room temperature, buffer B D described in 350 μ L is added, it is fully reverse
Brief centrifugation after mixing, obtains the second mixed liquor for including a certain amount of flocculent deposit；
Step 5：Second mixed liquor and the flocculent deposit are added into the adsorption column in the lump, and the adsorption column is put
Enter in the collecting pipe, in centrifuging 30sec under 12000rpm, take out the adsorption column, outwell the waste liquid in the collecting pipe, and
The adsorption column is put back into the collecting pipe；
Step 6：Buffer solution GDB described in 500 μ L is added into the adsorption column, in centrifuging 30sec under 12000rpm, is taken
Go out the adsorption column, outwell the waste liquid in the collecting pipe, and the adsorption column is put back into the collecting pipe；
Step 7：Rinsing liquid PWB described in 600 μ L is added into the adsorption column, in centrifuging 30sec under 12000rpm, is taken
Go out the adsorption column, outwell the waste liquid in the collecting pipe, and the adsorption column is put back into the collecting pipe；
Step 8：Rinsing liquid PWB described in 550 μ L is added into the adsorption column, in centrifuging 2.5min under 12000rpm, is taken
Go out the adsorption column, outwell the waste liquid in the collecting pipe, the adsorption column is inserted in the centrifuge tube；
Step 9：The lid of the adsorption column is opened, 2min is placed at room temperature；
Step 10：Elution buffer TB, room temperature described in 200 μ L is vacantly added dropwise in the adsorbed film centre position of the adsorption column
Place after 2min, in centrifuging 2min under 12000rpm, detection sample is obtained in the centrifuge tube.
Compared to prior art, the poba gene group DNA extraction method that the present invention is provided has the advantages that：
First, it is described using the blood sample is instilled in the step 2 of the poba gene group DNA extraction method
Mode in premixed liquid, solves the problem of often plus once the Proteinase K just needs to change a pipette tips, effectively reduces rifle
The consumption of head, alleviates the labor intensity of operating personnel.
2nd, in the step 4 of the poba gene group DNA extraction method, the buffer B D and first mixing
After the reverse mixing of liquid, also carrying out a brief centrifugation makes the two fully combine, and accelerates mixing time, prevents what is remained by lid
Liquid and cause the cross pollution between sample, and in the step 5 and subsequent step can obtain preferably separation or rinse imitate
Really.
3rd, in the step 8 of the poba gene group DNA extraction method, the use of the rinsing liquid PWB is suitably reduced
Amount, and extend centrifugation time, rinsing and centrifugally operated that originally need to repeatedly is reduced to the step 7 and the step 8
Two steps, and the purity of the detection sample is not influenceed；
Due to the reduction of the rinsing liquid PWB consumptions, the rinsing liquid will not be infected with again when taking out the adsorption column
PWB, without the operation individually once centrifuged again, simplifies operating procedure, shortens the extraction operating time, and to a certain degree
Improve the detection sample purity.
Embodiments of the invention are the foregoing is only, are not intended to limit the scope of the invention, it is every to utilize this hair
Equivalent structure or equivalent flow conversion that bright description is made, or directly or indirectly it is used in other related technology necks
Within domain, the scope of patent protection for being similarly included in the present invention.
Claims (4)
1. a kind of poba gene group DNA extraction method, poba gene group need to be used with the poba gene group DNA extraction method
DNA extraction kit, the poba gene group DNA extraction kit includes following components：
Buffer solution GB, buffer B D, buffer solution GDB, rinsing liquid PWB, elution buffer TB, Proteinase K, adsorption column, collecting pipe,
Centrifuge tube；
Characterized in that, the poba gene group DNA extraction method comprises the following steps：
Step 1：Extract 200 μ L blood sample；
Step 2：Proteinase K described in buffer solution GB described in 200 μ L and 20 μ L is combined as premixed liquid, the blood sample is added
Into the premixed liquid, fully vibration is mixed, and obtains the first mixed liquor；
Step 3：First mixed liquor at 56 DEG C in placing certain time, while reverse mix, until first mixed liquor
It is changed into clear liquid；
Step 4：First mixed liquor is placed into 2-5min at room temperature, buffer B D described in 350 μ L is added, it is fully reverse mixed
Even rear brief centrifugation, obtains the second mixed liquor；
Step 5：Second mixed liquor is added into the adsorption column, and the adsorption column is inserted in the collecting pipe, in
30sec is centrifuged under 12000rpm, the adsorption column is taken out, outwells the waste liquid in the collecting pipe, and the adsorption column is put back to
The collecting pipe；
Step 6：Buffer solution GDB described in 500 μ L is added into the adsorption column, in centrifuging 30sec under 12000rpm, takes out described
Adsorption column, outwells the waste liquid in the collecting pipe, and the adsorption column is put back into the collecting pipe；
Step 7：Rinsing liquid PWB described in 600 μ L is added into the adsorption column, in centrifuging 30sec under 12000rpm, takes out described
Adsorption column, outwells the waste liquid in the collecting pipe, and the adsorption column is put back into the collecting pipe；
Step 8：Rinsing liquid PWB described in 550 μ L is added into the adsorption column, in centrifuging 2.5min under 12000rpm, institute is taken out
Adsorption column is stated, the waste liquid in the collecting pipe is outwelled, the adsorption column is inserted in the centrifuge tube；
Step 9：The lid of the adsorption column is opened, 2min is placed at room temperature；
Step 10：Elution buffer TB, room temperature described in 50-200 μ L is vacantly added dropwise in the adsorbed film centre position of the adsorption column
Place after 2min, in centrifuging 2min under 12000rpm, detection sample is obtained in the centrifuge tube.
2. poba gene group DNA extraction method according to claim 1, it is characterised in that：The poba gene group DNA is carried
Kit is taken also to include buffer solution GS；
In the step one, when the amount of blood sample is less than 200 μ L, the buffer solution GS to 200 μ L is supplemented.
3. poba gene group DNA extraction method according to claim 2, it is characterised in that：The poba gene group DNA is carried
Kit is taken also to include cell pyrolysis liquid CL；
In the step one, when the amount of blood sample, which is more than 200 μ L, is less than 1mL, the step one is specifically divided into following steps：
Step 1.1：The cell pyrolysis liquid CL of the 1-2.5 times of blood sample volume, top are added in the blood sample
After mixing, in centrifuging 1min under 10000rpm, supernatant liquor is sucked, nucleus precipitation is left；
Step 1.2：Buffer solution GS described in 200 μ L is added in nucleus precipitation.
4. poba gene group DNA extraction method according to claim 1, it is characterised in that：Second described in the step 4
A certain amount of flocculent deposit is included in mixed liquor；
To in the lump it be added into the adsorption column comprising second mixed liquor including the flocculent deposit in the step 5.
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| CN201710473677.0A CN107245486A (en) | 2017-06-21 | 2017-06-21 | Poba gene group DNA extraction method |
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| CN201710473677.0A CN107245486A (en) | 2017-06-21 | 2017-06-21 | Poba gene group DNA extraction method |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102146374A (en) * | 2011-01-27 | 2011-08-10 | 四川农业大学 | Cell lysis solution for extracting animal DNA, kit and method |
| CN102154264A (en) * | 2011-01-25 | 2011-08-17 | 天根生化科技（北京）有限公司 | Method for rapidly extracting total ribonucleic acid from blood |
| CN102321613A (en) * | 2011-09-05 | 2012-01-18 | 吴剑 | Extraction method of clotting genomic DNA (deoxyribonucleic acid) |
| CN104017800A (en) * | 2014-06-20 | 2014-09-03 | 益百尚(北京)生物技术有限责任公司 | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof |
-
2017
- 2017-06-21 CN CN201710473677.0A patent/CN107245486A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154264A (en) * | 2011-01-25 | 2011-08-17 | 天根生化科技（北京）有限公司 | Method for rapidly extracting total ribonucleic acid from blood |
| CN102146374A (en) * | 2011-01-27 | 2011-08-10 | 四川农业大学 | Cell lysis solution for extracting animal DNA, kit and method |
| CN102321613A (en) * | 2011-09-05 | 2012-01-18 | 吴剑 | Extraction method of clotting genomic DNA (deoxyribonucleic acid) |
| CN104017800A (en) * | 2014-06-20 | 2014-09-03 | 益百尚(北京)生物技术有限责任公司 | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof |
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Address after: 410205 Hunan province Changsha high tech Zone Lu Tin Road No. 28 Minmetals Lugu science and Technology Industrial Park building D2 Applicant after: Changsha Jinyu medical laboratory Co., Ltd Address before: 410205 Hunan province Changsha high tech Zone Lu Tin Road No. 28 Minmetals Lugu science and Technology Industrial Park building D2 Applicant before: CHANGSHA KINGMED MEDICAL DIAGNOSTICS INSTITUTE Co.,Ltd. |
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Application publication date: 20171013 |