CN105713903A - Method for extracting DNA of unibract fritillary bulbs - Google Patents
Method for extracting DNA of unibract fritillary bulbs Download PDFInfo
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- CN105713903A CN105713903A CN201610234966.0A CN201610234966A CN105713903A CN 105713903 A CN105713903 A CN 105713903A CN 201610234966 A CN201610234966 A CN 201610234966A CN 105713903 A CN105713903 A CN 105713903A
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- microliters
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/1013—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 using magnetic beads
Abstract
The invention provides a method for extracting DNA of unibract fritillary bulbs, aims at providing a method which is high in specificity, high in enrichment efficiency and relatively easy to operate, and belongs to the technical field of chemical detection.The method sequentially comprises the following steps that 1, a sample is pretreated; 2, the pretreated sample is taken, 400 microliters of a splitting buffer solution and 5 microliters of molten ribonuclease A are added into the pretreated sample, and the materials are oscillated and mixed to be uniform; the materials are put in a metal bath at 70 DEG C for 10-30 minutes and taken out to be oscillated and mixed to be uniform for several times in the process; centrifuging at the revolution number of 5000 is performed for 10 minutes, and supernatant is taken to be extracted; a 96-site deep-hole plate in a kit is taken out, 300 microliters of the split sample, 600 microliters of a ligation buffer solution and a 60 microliters of magnetic beads are added in A-row holes, 500 microliters of a first cleaning buffer solution is added in F-row holes, 550 microliters of a second cleaning buffer solution is added in G-row holes, and 550 microliters of a third cleaning buffer solution is added in H-row holes; an eluting strip in the kit is taken out, and 100 microliters of an eluting buffer solution is added; the mixed 96-site deep-hole plate and eluting strip are put in a nucleic acid extractor for DNA extraction.
Description
Technical field
The invention provides the method for DNA that extracts Bulbus Fritillariae Cirrhosae a kind of, specifically, is one Plant the method for extracting Bulbus Fritillariae Cirrhosae DNA; Belong to chemical detection technique field.
Background technology
Bulbus Fritillariae Cirrhosae is mainly distributed in Sichuan, Yunnan, the ground such as Tibet, quality is more excellent, for lily section's plant Bulbus Fritillariae Cirrhosae, rib sand, the bulb of Wu Hua etc., there are clearing heat and moistening lung, preventing phlegm from forming and stopping coughing effect, medical value is higher. the discriminating of traditional Bulbus Fritillariae Cirrhosae, it is mainly proterties, micro-mirror not, spectroscopic methodology, because Bulbus Fritillariae kind on market is more, also can be because of between same breed for differing in medicine source plant resource source, planting environment, there is suitable mixing in the factor such as the place of production and processing assorted degree, this just makes experimenter must possess higher medicinal material identification of experience. along with molecule is raw the development that thing learns a skill, make the molecular genetic marker discriminating of Chinese medicine become possibility. at present on document, existing PCR (PCR) method is differentiated the relevant report of Bulbus Fritillariae Cirrhosae medicinal material road.
" Chinese pharmacopoeia " 2015 editions introduced PCR-restriction enzyme length polymorphism discrimination method to Bulbus Fritillariae Cirrhosae, but conventional PCR exists need be in conjunction with electrophoretic analysis, cannot detect in real time amplified reaction, and the shortcoming such as the toxic property of EB reagent.
Summary of the invention
For above-mentioned deficiency, the object of this invention is to provide and a kind ofly extract the DNA in Bulbus Fritillariae Cirrhosae by paramagnetic particle method, this extracting method specificity is strong, bioaccumulation efficiency is high, operation is relatively simple.
A method of extracting Bulbus Fritillariae Cirrhosae product DNA, comprises the steps: successively
1) pre-treatment sample
Get 1g sample, use mortar pulverize, then precision take 20mg to 2ml sterilizing from In core barrel;
2) extraction of DNA
The cracking of sample: get the sample that pre-treatment is good, add 400 μ L lysis buffers and The ribonuclease A 5 μ L that melt, vibration mixes; Be placed in 70 DEG C of metal bath 10-30 Minute, take out during this time vibration and mix for several times; 5000 leave the heart 10 minutes, get supernatant and carry Get; Take out 96 deep-well plates in kit, in capable lysate sample 300 μ that add respectively of A L, connects buffer solution 600 μ L, magnetic bead 60 μ L; Capable cleaning buffer solution I 500 μ that add of F L; The capable cleaning buffer solution II 550 μ L that add of G; Capable cleaning buffer solution III 550 μ that add of H L; Take out wash-out bar in kit, add elution buffer 100 μ L; By mixed 96 Position deep-well plates and wash-out bar, be placed in instrument for extracting nucleic acid, carries out DNA extraction.
Compared with prior art, because extracting mainly with reagent method, extracts traditional DNA Chang Yin Step is many, and often extraction efficiency is not high, to affect result accuracy; The present invention adopts magnetic bead Method is carried out DNA extraction, has the spies such as specificity is strong, bioaccumulation efficiency is high, operation is relatively simple Point.
Detailed description of the invention
Below in conjunction with detailed description of the invention; claim of the present invention is described in further detail; but do not form any limitation of the invention, the amendment of any limited number of time making at the claims in the present invention protection domain, still within claim protection domain of the present invention.
Embodiment 1
1. material and instrument
1.1 medicinal material
1.1.1 Bulbus Fritillariae Cirrhosae control medicinal material (National Institute for Food and Drugs Control, lot number: 121000-200405) Bulbus Fritillariae Cirrhosae 1,2 derives from Hui nationality's Chinese Medicinal Materials Markets, Bulbus Fritillariae Cirrhosae 3,4 and 5, fritillary bulb, fritillaria thunbergii and the rhizoma bolbostemmae derive from Guangxi Yulin Chinese Medicinal Materials Markets, and confirm through Morphological Identification.
1.1.2 reagent and primer paramagnetic particle method DNA of plants extract kit (power & light company of the U.S., lot number: 00LSKFR804096F25N009), SuperReal fluorescent quantitation premix reagent enhanced edition (Tian Gen biochemical technology Co., Ltd, lot number: 03120), Bulbus Fritillariae Cirrhosae primer (invitrogen company, lot number: NSO_2954885, sequence is:
5 ' CGTAACAAGGTTTCCGTAGGTGAA3 ' and
5’GCTACGTTCTTCATCGAT3’)。
The every pipe of described primer adds ultra-pure water 0.29ml and dilutes, and vortex vibration mixes it is fully dissolved, for subsequent use in-20 DEG C of Refrigerator stores.
The percentage concentration arriving involved in the present invention, does not specify, liquid is volumetric concentration, the finger mass concentration that solute is solid.
1.2 instrument XA205DU electronic balances (German Mei Teletuo benefit company), Minispin table model high speed centrifuge (German Eppendorf), MINIB-100F constant-temperature metal bath (Hangzhou meter Ou Instrument Ltd.), LightCycler96 quantitative real time PCR Instrument (Roche Holding Ag of Switzerland), Thermo Fisher Duo instrument for extracting nucleic acid (power & light company of the U.S.).
1g sample is got in the pre-treatment of 2.1 samples, uses mortar pulverize, then precision takes in 20mg to 2ml sterilizing centrifuge tube.
The extraction of 2.2DNA
2.2.1 the cracking of sample: get the sample that pre-treatment is good, the RNase A 5 μ L that add 400 μ L LysisBuffer and melted, vibration mixes. and be placed in 70 DEG C of metal bath 10-30 minute, taking-up vibration during this time mixes .5000 for several times and leaves the heart 10 minutes, gets supernatant and extracts.
2.2.2 paramagnetic particle method extracts sample DNA: take out 96 deep-well plates in kit, in A Row adds respectively lysate sample 300 μ L, Binding Buffer 600 μ L, magnetic bead 60 μ L; The capable cleaning buffer solution I 500 μ L that add of F; Capable cleaning buffer solution II 550 μ that add of G L; H is capable, and the cleaning buffer solution III 550 μ L. that add take out wash-out bar in kit, add and wash De-buffer solution 100 μ L., by mixed 96 deep-well plates and wash-out bar, are placed in nucleic acid and carry Get instrument, carry out DNA extraction.
For the material that proves that the present invention extracts, the present invention also provides the detection method of this primer:
In the every pipe PCR of the preparation pipe of PCR reactant liquor, add respectively 2x SuperReal PreMix Plus (with SYBR Green I) 10 μ L, the each 0.5 μ L of positive and negative primer and RNase-Free ddH2O 7 μ L.
PCR adds, in the above-mentioned PCR pipe containing reactant liquor, the DNA that 2 μ L extract,
Beat PCR pipe box gently, make reactant liquor and DNA mix, and through 3000 leave the heart 3～
10 seconds, carry out PCR reaction immediately.
Quantitative real time PCR Instrument arranges
Method arranges: detecting pattern is fluorescence channel, reaction cumulative volume is 20 μ L, SYBR Green I is selected in dyeing, selects successively " Preincubation " (denaturation) and " 3Step Amplification " (three step amplifications), selects Single to collect fluorescence.
QPCR response parameter arranges by table 1:
Table 1qPCR response parameter is arranged
Data process
By on data syn-chronization to computer, select " qualitative detection " that sample is analyzed.
Result
The results are shown in Table 2, wherein Bulbus Fritillariae Cirrhosae control medicinal material, Bulbus Fritillariae Cirrhosae 1,2,3,4,5 and Positive control Cq value all < 30, and amplification curve has an obvious Exponential growth stage, thus can determine that into Positive; And other Bulbus Fritillariae Uninbracteatae class and negative control are all without Cq value, and curve is straight line, Therefore be negative.Through test, sample result stable and consistent are repeated several times, show the method pair The discriminating accuracy rate of Bulbus Fritillariae Cirrhosae is high, specificity is strong.
The qPCR result of sample
Claims (1)
1. the method extracting Bulbus Fritillariae Cirrhosae product DNA, it is characterised in that under including successively states step:
1) pre-treatment sample
Get 1g sample, use mortar pulverize, then precision take 20mg to 2ml sterilizing from In heart pipe;
2) extraction of DNA
Get the sample that pre-treatment is good, the ribose core that adds 400 μ L lysis buffers and melted Acid enzyme A 5 μ L, vibration mixing; Be placed in 70 DEG C of metal bath 10-30 minute, get during this time Go out vibration mixing for several times; 5000 leave the heart 10 minutes, take supernatant and extract; Take out reagent 96 deep-well plates in box, are separately added into lysate sample 300 μ L in A row, connect buffering Liquid 600 μ L, magnetic bead 60 μ L; F row adds cleaning buffer solution I 500 μ L; G row adds Cleaning buffer solution II 550 μ L; H row adds cleaning buffer solution III 550 μ L; Take out test kit Middle eluting bar, adds elution buffer 100 μ L; By mixed 96 deep-well plates and washing De-bar, is placed in instrument for extracting nucleic acid, carries out DNA extraction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610234966.0A CN105713903A (en) | 2016-04-15 | 2016-04-15 | Method for extracting DNA of unibract fritillary bulbs |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610234966.0A CN105713903A (en) | 2016-04-15 | 2016-04-15 | Method for extracting DNA of unibract fritillary bulbs |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111575273A (en) * | 2020-04-08 | 2020-08-25 | 康美华大基因技术有限公司 | Traditional Chinese medicine fritillary DNA extraction reagent and extraction method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792757A (en) * | 2010-03-30 | 2010-08-04 | 上海鼎国生物技术有限公司 | Kit for separating genome DNA by using magnetic balls and application thereof |
| CN104450684A (en) * | 2014-12-26 | 2015-03-25 | 南京中科神光科技有限公司 | Kit for extracting nucleic acid from bacteria by using paramagnetic particle method and extracting method |
-
2016
- 2016-04-15 CN CN201610234966.0A patent/CN105713903A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792757A (en) * | 2010-03-30 | 2010-08-04 | 上海鼎国生物技术有限公司 | Kit for separating genome DNA by using magnetic balls and application thereof |
| CN104450684A (en) * | 2014-12-26 | 2015-03-25 | 南京中科神光科技有限公司 | Kit for extracting nucleic acid from bacteria by using paramagnetic particle method and extracting method |
Non-Patent Citations (1)
| Title |
|---|
| 陈丽丽: "玉米基因组DNA提取测定及转植酸酶基因检测方法的研究", 《中国优秀硕士论文全文期刊库农业科技辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111575273A (en) * | 2020-04-08 | 2020-08-25 | 康美华大基因技术有限公司 | Traditional Chinese medicine fritillary DNA extraction reagent and extraction method |
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