CN101235350A - Soil DNA sample preparation kit and preparation method thereof - Google Patents

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

A kind of soil DNA sample preparation reagents box and preparation method thereof

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 to use soil bacteria diversity analysis method the most widely, and the DNA specimen 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 the purifying process complexity, and approach is various, and the amplification difficulty is big, 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 specimen preparation complex process, difficulty are big, 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) 10mmol/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 (pure product); It is Sephadex G752% (mass volume ratio) that the proportioning of suspension V is formed; Pickling glass pearl 0.5g, the about 0.2mm of diameter; 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 the ambient soil sample be used to test;

B) agricultural land soil that will be used for step a) obtains the genomic dna of all microorganisms in the soil after the cell pyrolysis liquid cracking;

C) behind the genomic dna purifying that step b) is obtained as the template of pcr amplification;

D) it is right to select generally to be used for the universal primer of prokaryotic organism 16SrRNA (16S rRNA) gene amplification;

E) end at the right forward primer of the universal primer of step d) selection adds the GC hairpin structure;

F) the PCR reaction conditions according to design carries out pcr amplification;

G) the described pcr amplification product of step f) is standby as sample retention.

By the test kit that technical solution of the present invention designs, it is stable to have easy to use, result, the advantage that can save user's time and cost effectively.

Description of drawings

Fig. 1 forms synoptic diagram for test kit;

Fig. 2 is a test kit operation steps synoptic 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 (as the water content height, centrifugal 2 minutes of 10000g, taking precipitate is standby) 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:, 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 if solution II is solidified;

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 tight lid (annotating:, add 10ul sterile liquid paraffin again) if used PCR instrument does not have the heat lid;

8. PCR pipe (H) is put into the PCR instrument, the temperature of reaction circulation is provided with as follows: (1) 94 ℃ of sex change 10 minutes; (2) below 10 circulations, 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. it is 4. standby to obtain the DNA sample ,-20 ℃ of preservations.

Claims (6)

1. soil DNA sample preparation reagents box, comprise box body and dividing plate, it is characterized in that: the reagent bottle A that fills 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.

2. a kind of soil DNA sample preparation reagents box according to claim 1, it is characterized in that: 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; Solution III is the ammonium acetate of 5mol/l; Solution IV is a Virahol; It is that mass volume ratio is 2% Sephadex G75 that the proportioning of suspension V is formed; Pickling glass pearl 0.5g, the about 0.2mm of diameter; 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.

3. 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.

4. 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.

5. 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.

6. the soil DNA sample preparation steps is as follows:

A) select the ambient soil sample be used to test;

B) will be used for steps A) agricultural land soil 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) it is right to select generally to be used for the universal primer of prokaryotic organism 16SrRNA (16S rRNA) gene amplification;

E) at step 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 standby as sample retention.

Images