CN101235350B - Soil DNA sample preparation kit and preparation method thereof - Google Patents
Soil DNA sample preparation kit and preparation method thereof Download PDFInfo
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- CN101235350B CN101235350B CN 200710037850 CN200710037850A CN101235350B CN 101235350 B CN101235350 B CN 101235350B CN 200710037850 CN200710037850 CN 200710037850 CN 200710037850 A CN200710037850 A CN 200710037850A CN 101235350 B CN101235350 B CN 101235350B
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Abstract
The invention discloses a soil DNA sample preparation reagent box and a manufacturing method, the reagent box comprises a box body and a baffle board, a reagent bottle A which is filled with solution I, a reagent bottle B which is filled with solution II, a reagent bottle C which is filled with solution III, a reagent bottle D which is filled with solution IV, a reagent bottle E which is filled with suspension liquid, a centrifuge tube F which is filled with acid cleaning glass bead, a mini filter G and a PCR(chain type polyase reaction) pipe H. A reagent box which is designed through the technical proposal of the invention has the advantages of simple employment and stable result, which can effectively save time and cost of users.
Description
Technical field
The present invention relates to a kind of soil DNA sample preparation reagents box and preparation method thereof, be used for the preparation that denaturing gradient gel electrophoresis is analyzed the multifarious DNA sample of soil bacteria, belong to the DNA analysis technical field.
Background technology
At present, denaturing gradient gel electrophoresis is the most widely used soil bacteria diversity analysis method, and the DNA sample preparation process that is used for denaturing gradient gel electrophoresis comprises the amplification of extraction, purifying and the target dna of the total DNA of soil.Yet soil DNA extracts and purifying process is complicated, and approach is various, and the amplification difficulty is large, and preparation process wastes time and energy, and result comparison is poor.
Summary of the invention
Goal of the invention of the present invention is for a kind of soil DNA sample preparation reagents box and preparation method thereof is provided, to overcome the problem that existing DNA sample preparation complex process, difficulty are large, waste time and energy.
Goal of the invention of the present invention can be achieved through the following technical solutions.
A kind of soil DNA sample preparation reagents box comprises box body and dividing plate; Fill the reagent bottle A of solution I, fill the reagent bottle B of solution II, fill the reagent bottle C of solution III, fill the reagent bottle D of solution IV, fill the reagent bottle E of suspension V, include the centrifuge tube F of pickling glass pearl, microfilter G, PCR (chain polymerization enzyme reaction) manages H.
Wherein, solution I is the phosphate buffered saline buffer of 120mmol/l; The proportioning of solution II consists of TrisHCl (Tutofusin tris hydrochloric acid) 10mmo l/l, EDTA (ethylenediamine tetraacetic acid (EDTA)) 1mmol/l, CTAB (cetyl trimethylammonium bromide) 0.2%, pH8.0; Solution III is the ammonium acetate of 5mol/l; Solution IV is Virahol (sterling); It is Sephadex G752% (mass volume ratio) that the proportioning of suspension V forms; Pickling glass pearl 0.5g, diameter 0.2mm; The volume of PCR pipe H is 200 μ l, in fill each 0.25 μ mol of dna primer, Taq enzyme 2U, each 200 μ mol of 4 kinds of dNTP (thymus nucleic acid), 1 * PCR reaction buffer 100ul, lyophilize gained.
Centrifuge tube F is the centrifuge tube of 2ml.
All reagent and consumptive material are all through aseptically process.
Described Virahol is kept in the brown reagent bottle.
The soil DNA sample preparation steps is as follows:
A) select for the ambient soil sample of testing;
B) will for step agricultural land soil a) after the cell pyrolysis liquid cracking, obtain the genomic dna of all microorganisms in the soil;
C) with step b) behind the genomic dna purifying that obtains as the template of pcr amplification;
D) select the general universal primer that is used for prokaryotic organism 16SrRNA (16S rRNA) gene amplification pair;
E) in steps d) end of the right forward primer of the universal primer selected adds the GC hairpin structure;
F) the PCR reaction conditions according to design carries out pcr amplification;
G) with step f) described pcr amplification product is for subsequent use as sample retention.
By the test kit that technical solution of the present invention designs, has easy to use, result stable, the advantage that can effectively save user's time and cost.
Description of drawings
Fig. 1 is that test kit forms schematic diagram;
Fig. 2 is test kit operation steps schematic diagram.
Embodiment
Further set forth technical characterstic of the present invention below in conjunction with accompanying drawing and specific embodiment.
1. take by weighing pending drying disperse (high such as water content, centrifugal 2 minutes of 10000g, taking precipitate is for subsequent use) pedotheque 0.5g to centrifuge tube F, add 1ml solution I (A), slight vortex concussion mixing, the centrifugal 1～2min of 2000g, remove supernatant liquor, repetitive operation once;
2. get 800ul solution II (B) (annotating: if solution II is solidified, please place 60 ℃ of water-bath heating for dissolving) to centrifuge tube F, violent vortex concussion 2～5 minutes, centrifugal 1～2 minute of 10000g;
3. pipette supernatant liquor 400ul to the 1.5ml centrifuge tube (providing for oneself) of cleaning sterile, add 400ul solution III (C), 4 ℃ are incubated 20 minutes, centrifugal 5～10 minutes of 10000g;
4. draw supernatant liquor as far as possible and to the 2ml centrifuge tube, (provide for oneself), add 800ul solution IV (D), put upside down and mix ,-20 ℃ of insulations 20 minutes, centrifugal 5～10 minutes of 10000g;
5. abandon supernatant liquor, add 200ul TE (Tris-EDTA) solution (or sterilized water) suspended sediment, get solution 2.;
6. pipette solution 2. to gel-filtration column 1. in, 3. centrifugal 1 minute of 1000g obtains filtrate;
7. add the aseptic ultrapure water of 98ul to PCR pipe (H), add 2ul solution 3., cover tightly lid (annotating: if used PCR instrument does not have the heat lid, add again 10ul sterile liquid paraffin);
8. PCR pipe (H) is put into the PCR instrument, the temperature of reaction circulation arranges as follows: (1) 94 ℃ of sex change 10 minutes; (2) be 10 circulations below, 94 ℃ of sex change 1 minute, 65 ℃～55 ℃ renaturation 1 minute, each circulation of renaturation temperature reduces by 1 ℃, and 72 ℃ prolong 1 minute; (3) be 20 circulations below, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute; (4) 72 ℃ prolong 8 minutes, 4 ℃ of insulations.
9. obtain the DNA sample 4. for subsequent use ,-20 ℃ of preservations.
Claims (4)
1. soil DNA sample preparation reagents box, comprise box body and dividing plate, it is characterized in that: the reagent bottle A that fills the solution I, fill the reagent bottle B of solution II, fill the reagent bottle C of solution III, fill the reagent bottle D of solution IV, fill the reagent bottle E of suspension V, include the centrifuge tube F of pickling glass pearl, microfilter G, the enzyme reaction of PCR(chain polymerization) pipe H;
Described solution I is the phosphate buffered saline buffer of 120mmol/l; The proportioning of solution II consists of TrisHCl 10mmol/l, EDTA 1mmol/l, and CTAB 0.2%, pH8.0; The solution III is the ammonium acetate of 5mol/l; The solution IV is Virahol; It is that mass volume ratio is 2% Sephadex G75 that the proportioning of suspension V forms; Pickling glass pearl 0.5g, diameter 0.2mm; The volume of PCR pipe H is 200 μ l, in fill each 0.25 μ mol of dna primer, Taq enzyme 2U, 4 kinds of each 200 μ mol of dNTP, 1 * PCR reaction buffer 100ul, lyophilize gained;
Described dna primer is the universal primer that is used for prokaryotic organism 16SrRNA gene amplification pair.
2. a kind of soil DNA sample preparation reagents box according to claim 1, it is characterized in that: centrifuge tube F is the centrifuge tube of 2ml.
3. a kind of soil DNA sample preparation reagents box according to claim 1, it is characterized in that: all reagent and consumptive material are all through aseptically process.
4. a kind of soil DNA sample preparation reagents box according to claim 1, it is characterized in that: described Virahol is kept in the brown reagent bottle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710037850 CN101235350B (en) | 2007-03-06 | 2007-03-06 | Soil DNA sample preparation kit and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710037850 CN101235350B (en) | 2007-03-06 | 2007-03-06 | Soil DNA sample preparation kit and preparation method thereof |
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| Publication Number | Publication Date |
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| CN101235350A CN101235350A (en) | 2008-08-06 |
| CN101235350B true CN101235350B (en) | 2013-02-27 |
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| CN 200710037850 Expired - Fee Related CN101235350B (en) | 2007-03-06 | 2007-03-06 | Soil DNA sample preparation kit and preparation method thereof |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105925563A (en) * | 2016-05-13 | 2016-09-07 | 李丙亮 | Method for preparing stationary-phase nucleic acid reagent |
| CN113416629B (en) * | 2021-06-03 | 2023-09-22 | 中南大学湘雅三医院 | Tumor chemotherapeutic drug sensitivity detection kit and detection method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1456684A (en) * | 2003-03-26 | 2003-11-19 | 中国科学院生态环境研究中心 | Induction design plan for PCR-DGGE research on environment microorgan population |
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2007
- 2007-03-06 CN CN 200710037850 patent/CN101235350B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1456684A (en) * | 2003-03-26 | 2003-11-19 | 中国科学院生态环境研究中心 | Induction design plan for PCR-DGGE research on environment microorgan population |
Non-Patent Citations (3)
| Title |
|---|
| 方光伟等.土壤宏基因组的提取及基于免培养技术分析细菌16S rDNA.《江西农业大学学报》.2005,第27卷(第4期),第505-507页. * |
| 赵勇等.土壤微生物分子生态学研究中总DNA的提取.《农业环境科学学报》.2005,第24卷(第5期),第854-860页. * |
| 魏志琴等.土壤微生物DNA提取方法研究进展.《遵义师范学院学报》.2006,第8卷(第4期),第53-56页. * |
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| CN101235350A (en) | 2008-08-06 |
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