CN115011588B - Bacterial genome DNA extraction kit and using method thereof - Google Patents

Bacterial genome DNA extraction kit and using method thereof Download PDF

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CN115011588B
CN115011588B CN202210610398.5A CN202210610398A CN115011588B CN 115011588 B CN115011588 B CN 115011588B CN 202210610398 A CN202210610398 A CN 202210610398A CN 115011588 B CN115011588 B CN 115011588B
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dna
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adsorption column
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CN115011588A (en
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肖晓文
王文朋
李妍
东昱汝
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Tsingke Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

Abstract

The invention belongs to the technical field of biology, and discloses a bacterial genome DNA extraction kit and a bacterial genome DNA extraction method. The kit provided by the invention adopts a unique buffer solution system and a centrifugal column for specifically adsorbing DNA, can rapidly and efficiently extract genome DNA of various gram negative/positive bacteria within 1h, and can remove impurity proteins and other metabolic substances in cells to the maximum extent.

Description

Bacterial genome DNA extraction kit and using method thereof
Technical Field
The invention relates to the technical field of biology, in particular to a kit for extracting bacterial genome DNA and a use method thereof.
Background
Along with the development of biochemistry and molecular biology such as molecular cloning technology, PCR technology, genetic engineering, whole-gene DNA sequencing technology and the like, the research on biological DNA is more and more extensive and intensive. The extraction of genome DNA is often the first step of biological DNA research and is also the basis of the research, and the extraction quality of DNA directly determines the success or failure of downstream experiments.
Bacteria belong to prokaryotes, and have relatively simple cell structures and tough cell walls composed of peptidoglycan. The first step of bacterial DNA extraction requires the destruction of the cell wall and the release of the contents of nucleic acids and the like in the cells, and the current method for extracting bacterial genomic DNA mainly comprises the following steps: cell disruption, enzymatic, chemical, and the like, and DNA extraction. Wherein the mechanical method mainly utilizes mechanical acting force such as ultrasonic crushing, repeated freeze thawing, microbead vibration and the like to crush bacterial cell walls; the enzymatic method mainly comprises the steps of dissolving and crushing bacterial cell walls by biological enzymes such as lysozyme and the like; the chemical method is to extract protein impurities by combining organic solvents such as breaking bacteria and protein denaturation through chemical reagents such as strong alkali NaOH, surfactant SDS (sodium dodecyl sulfate), CTAB and the like, so as to obtain DNA with higher purity.
Genome research of bacteria is becoming more extensive and important, so that efficient, high quality extraction of their genome is also of great importance. The existing extraction modes of various bacterial genomes have different defects such as long time consumption, use of toxic reagents, low product purity and the like. Therefore, a kit for extracting bacterial genome needs to be developed, and the corresponding problem of bacterial genome extraction is effectively solved.
Disclosure of Invention
The invention aims to provide a kit for extracting bacterial genome DNA, which effectively solves the corresponding problem of bacterial genome extraction.
Thus, in a first aspect, the present invention provides a bacterial genomic DNA extraction kit comprising a suspension, a binding solution, a deproteinizing solution, a rinsing solution and an eluent,
the suspension comprises 1-2%, preferably 1.5% sarcosyl in a first Tris-EDTA buffer;
the binding solution comprises 2-5M, preferably 2.5M GuHCl and 20-60mM, preferably 40mM urea in sodium citrate buffer;
the deproteinized solution comprises 2-3M, preferably 2.5M GuHCl in a first Tris-HCl buffer, and ethanol with a final concentration of 30-60%, preferably 55% by volume;
the rinsing solution comprises adding ethanol with a final concentration of 75-80%, preferably 80% by volume, to a second Tris-HCl buffer;
the eluent includes a second Tris-EDTA buffer.
In one embodiment, the kit further comprises a DNA adsorption column and proteinase K.
In one embodiment, the DNA adsorption column is a silicon matrix membrane adsorption column.
In one embodiment, the pH of the first Tris-EDTA buffer is 7.5 to 8.5, preferably 8, including 20 to 100mM, preferably 25 to 50mM, tris-HCl and 10 to 20mM EDTA.
In one embodiment, the pH of the sodium citrate buffer is from 5.0 to 6.0, preferably from 5.0 to 5.5, more preferably from 5.0 to 5.05, at a concentration of 20 to 60mM, preferably 50mM.
In one embodiment, the pH of the first Tris-HCl buffer is between 6.8 and 7.5, preferably 7.0, at a concentration of between 20 and 50mM, preferably 30mM.
In one embodiment, the pH of the second Tris-HCl buffer is between 6.8 and 7.5, preferably 7.0, at a concentration of between 10 and 20mM, preferably 15mM.
In one embodiment, the pH of the second Tris-EDTA buffer is 7.5 to 8.5, preferably 8, including 5 to 20mM, preferably 10mM Tris-HCl and 0.1 to 10mM, preferably 1mM EDTA.
In a second aspect, the present invention provides a method of bacterial genomic DNA extraction comprising:
1) Breaking bacterial cells, degrading proteins and releasing bacterial genomic DNA from the bacterial sample;
2) Adding the binding solution of the invention for incubation;
3) Precipitating the DNA with ethanol;
4) Transferring the liquid of the precipitated DNA into an adsorption column for centrifugation, and removing liquid phase;
5) Adding the deproteinized liquid and absolute ethanol mixture of the invention into the adsorption column for centrifugation, and removing liquid phase;
6) Adding the mixture of the rinsing liquid and the absolute ethyl alcohol into the adsorption column for centrifugation, removing liquid phase, preferably repeating for 1-2 times, and then airing;
7) Eluting DNA from the adsorption column.
In one embodiment, in 1), the cell walls of gram-negative bacteria are disrupted with the suspension of the invention and the cell walls of gram-positive bacteria are disrupted with lysozyme.
In one embodiment, in 1), the protein is degraded with proteinase K.
In one embodiment, in 2), incubation is carried out at 56℃for 10-15 min.
In one embodiment, in 4) -6), centrifugation is performed at 12,000rpm (about 13,400 Xg) for 1min.
In one embodiment, in 7), the elution is performed with TE Buffer or sterile water, preferably at a temperature of 65 ℃.
The components in the kit are matched in a synergistic way, so that the excellent DNA extraction effect of the kit is realized. In the course of development, the inventors found that not all similar reagents perform well, such as suspensions, and if SLS is replaced with SDS, tween20, triton X-100 or other reagents such as guanidine hydrochloride, the problems of excessive extraction or RNA residue or poor amplification effect are caused, or the effects are also deteriorated when SDS and salts such as sodium chloride are used together. For example, if GuSCN, sodium acetate, NP-40, PEG-8000 or Tris-HCl, triton X-100, AEO-15 are used in the binding solution, the solution is flocculent, the extraction effect is poor or the effect is unstable. The deproteinizing solution has the main function of creating conditions of high salt and low pH, so that the nucleic acid is adsorbed on the silicon substrate film, and the protein is promoted to be dissolved in the deproteinizing solution and eluted from the silicon substrate film; however, improper deproteinization solution selection can cause problems such as salt residue and PCR inhibition; in the system of the invention, the salt of the deproteinized liquid is selected by comprehensively considering the components of the suspension and the binding liquid, and the ideal impurity removing effect can be achieved only by mutual matching, thus improving the purity of the product; for example, the use of sodium chloride or GuSCN or its combination with guanidine hydrochloride, or the addition of auxiliary components such as urea, triton X-100, may result in poor results. In addition, components can also be mutually influenced, for example, the components of the suspension and the binding solution need to be matched with each other to achieve a balance effect, so that bacterial cells can be effectively lysed, and if SDS is used in the suspension, the conditions of turbidity of the solution, blockage of a column and the like can occur after the binding solution is added. In addition, details such as the amount of each reagent and pH will also affect the results. For example, when the guanidine hydrochloride concentration is too high, the residual amount of RNA is significantly increased; guanidine hydrochloride affects the pH measurement and requires the pH of the binding solution to be adjusted by the pH of the sodium citrate buffer.
The kit and the method for extracting the bacterial genome DNA by utilizing the DNA adsorption column can be used for rapidly extracting the genome DNA of various gram-negative bacteria and positive bacteria, and ensure that the obtained genome DNA has higher purity and is compatible with downstream application.
Drawings
FIG. 1 shows gel electrophoresis patterns of bacterial genomic DNA extracted using different formulations of suspensions and binding solutions in combination;
FIG. 2 shows gel electrophoresis of a combination of suspension A and different binding solutions using extracted bacterial genomic DNA; wherein, (A) is a gel electrophoresis diagram of the extracted escherichia coli genome DNA, and (B) is a gel electrophoresis diagram of the extracted agrobacterium genome DNA;
FIG. 3 shows gel electrophoresis patterns of bacterial genomic DNA extracted using deproteinized solutions of different formulations; wherein, (A) is a gel electrophoresis diagram of the extracted escherichia coli genome DNA, and (B) is a gel electrophoresis diagram after PCR amplification by using different primers;
FIG. 4 shows gel electrophoresis patterns of bacterial genomic DNA extracted using deproteinized solutions of different formulations; wherein, (A) is a gel electrophoresis diagram of the extracted escherichia coli genome DNA, and (B) is a gel electrophoresis diagram after PCR amplification by using different primers;
FIG. 5 shows a gel electrophoresis pattern of bacterial genomic DNA extracted after adjustment of buffer in deproteinized solution;
FIG. 6 shows gel electrophoresis of genomic DNA extracted from different bacteria using the present kit and a commercially available kit.
Detailed Description
The present invention will be described in detail below. It is to be understood that the following description is intended to illustrate the invention by way of example only, and is not intended to limit the scope of the invention as defined by the appended claims. And, it is understood by those skilled in the art that the technical scheme of the present invention can be modified without departing from the spirit and gist of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided to better understand the present invention.
Where a range of values is provided, such as a range of concentrations, a range of percentages, or a range of ratios, it is to be understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of the range, and any other stated or intervening value in that stated range, is encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also included in the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.
The kit provided by the invention adopts a unique buffer solution system and a centrifugal column for specifically adsorbing DNA, can rapidly and efficiently extract genome DNA of various gram negative/positive bacteria within 1h, and can remove impurity proteins and other metabolic substances in cells to the maximum extent. The kit does not need to use phenol/chloroform extraction, does not need to carry out a time-consuming isopropanol or ethanol precipitation process, and is safe to operate. The genome DNA obtained by using the kit has good integrity and high purity, and can be directly used for experiments such as PCR, enzyme digestion, hybridization and the like.
Without wishing to be bound by any theory, the inventors believe that the kit of the invention achieves better bacterial DNA extraction results, i.e. good bacterial genomic DNA integrity and high purity, than conventional methods of the prior art by accurate reagent component loading sequences and accurate pH and concentration.
Examples
Example 1, reagent components and concentrations of the kit.
Formula 1:
suspension: tris-HCl (pH 8), 50mM; EDTA (pH 8), 10mM; sodium dodecyl sarcosinate (SLS), 1.5%.
Binding liquid: sodium citrate (pH 5.0-5.05), 50mM; guHCl,2.5M; urea, 40mM.
Deproteinized liquid: tris-HCl (pH 7), 30mM; guHCl,2.5M; ethanol, 55%.
Rinsing liquid: tris-HCl (pH 7), 15mM; ethanol, 80%.
Eluent (TE pH 8): tris-HCl (pH 8), 10mM; EDTA (pH 8), 1mM.
Formula 2:
suspension: tris-HCl (pH 8), 25mM; EDTA (pH 8), 10mM; sodium dodecyl sarcosinate (SLS), 1.5%.
Binding liquid: sodium citrate (pH 5.0), 50mM; guHCl,2.5M; urea, 40mM.
Deproteinized liquid: tris-HCl (pH 7), 30mM; guHCl,2.5M; ethanol, 55%.
Rinsing liquid: tris-HCl (pH 7), 15mM; ethanol, 80%.
Eluent (TE pH 8): tris-HCl (pH 8), 10mM; EDTA (pH 8), 1mM.
Formula 3:
suspension: tris-HCl (pH 8), 25mM; EDTA (pH 8), 10mM; sodium dodecyl sarcosinate (SLS), 1.5%.
Binding liquid: sodium citrate (pH 5.2), 50mM; guHCl,2.0M; urea, 20mM.
Deproteinized liquid: tris-HCl (pH 7.5), 50mM; guHCl,3M; ethanol, 60%.
Rinsing liquid: tris-HCl (pH 7.5), 10mM; ethanol, 80%.
Eluent (TE pH 8): tris-HCl (pH 8), 10mM; EDTA (pH 8), 1mM.
Formula 4:
suspension: tris-HCl (pH 8), 50mM; EDTA (pH 8), 10mM; sodium dodecyl sarcosinate (SLS), 1.5%.
Binding liquid: sodium citrate (pH 5.03), 50mM; guHCl,2.5M; urea, 40mM.
Deproteinized liquid: tris-HCl (pH 6.8), 20mM; guHCl,2.5M; ethanol, 45%.
Rinsing liquid: tris-HCl (pH 6.8), 20mM; ethanol, 75%.
Eluent (TE pH 8): tris-HCl (pH 8), 10mM; EDTA (pH 8), 1mM.
Formula 5:
suspension: tris-HCl (pH 8), 50mM; EDTA (pH 8), 20mM; sodium dodecyl sarcosinate (SLS), 1.0%.
Binding liquid: sodium citrate (pH 5.5), 40mM; guHCl,2.5M; urea, 30mM.
Deproteinized liquid: tris-HCl (pH 7), 30mM; guHCl,2.5M; ethanol, 55%.
Rinsing liquid: tris-HCl (pH 7), 15mM; ethanol, 80%.
Eluent (TE pH 8): tris-HCl (pH 8), 10mM; EDTA (pH 8), 1mM.
Example 2 bacterial genomic DNA extraction method procedure.
1. Sample preparation.
1.1 gram-negative bacteria.
1) 1-5 mL (not more than 1×10) of bacterial culture is taken 9 Individual cells) were placed in a centrifuge tube and centrifuged at 12,000rpm (13,400 Xg) for 1min, and the supernatant was aspirated as much as possible.
2) 200. Mu.L of the suspension of the present invention was added thereto, and the mixture was shaken until the cells were thoroughly suspended.
3) Optionally adding 5 mu L of RNase A, oscillating for 15s, and standing at room temperature for 5-15 min.
4) Add 20. Mu.L of proteinase K solution to the tube and mix well.
1.2 gram-positive bacteria.
1) 1-5 mL (not more than 1×10) of bacterial culture is taken 9 Individual cells) were placed in a centrifuge tube and centrifuged at 12,000rpm (13,400 Xg) for 1min, and the supernatant was aspirated as much as possible.
2) 180. Mu.L of lysozyme was added and the cells were shaken until they were thoroughly suspended and incubated at 37℃for 30min or more. The concentration of the lysozyme is 20mg/mL, and the preparation method of the lysozyme buffer solution comprises the following steps: 20mM Tris, pH8.0;2mM Na 2 -EDTA；1.2％Triton X-100。
3) Optionally adding 5 mu L of RNase A, oscillating for 15s, and standing at room temperature for 5-15 min.
4) Add 20. Mu.L of proteinase K solution to the tube and mix well.
2. 220 mu L of the binding solution of the invention is added, mixed evenly by shaking, and incubated for 10-15 min at 56 ℃.
3. 220. Mu.L of absolute ethanol is added and mixed by shaking, and flocculent precipitate may appear at this time.
4. The mixed solution obtained in the previous step was transferred to an adsorption column loaded in a collection tube, centrifuged at 12,000rpm (13,400 Xg) for 1min, the waste liquid was poured, and the adsorption column was returned to the collection tube. The adsorption column may be a silicon matrix membrane comprising the advantages of: the method is simple to operate, changes the nucleic acid separation and purification process into simple filtration operation, has low operation requirement and good repeatability; the purification effect is good, and the yield is high; high safety and no need of contacting with great amount of toxic reagent.
5. Absolute ethanol was added to 500. Mu.L of deproteinized solution, the final concentration of ethanol by volume was 55%, and the solution was centrifuged at 12,000rpm (13,400 Xg) for 1min, and the waste liquid was discarded.
6. Absolute ethanol was added to 600. Mu.L of the rinse solution, the final concentration of ethanol by volume was 55%, and the solution was centrifuged at 12,000rpm (13,400 Xg) for 1min, and the waste solution was discarded, preferably repeated once.
7. The column was returned to the collection tube, centrifuged at 12,000rpm (13,400 Xg) for 2min, the waste liquid was decanted off, and the column was left to stand at room temperature for several minutes with the column open to allow for adequate air drying.
8. The adsorption column is taken out, placed into a clean 1.5mL centrifuge tube, 50-100 mu L TE Buffer or sterilized water is dripped in the center of the adsorption film for eluting (preferably 65 ℃), and placed for 2-5 min at room temperature, and centrifuged at 12,000rpm (13,400 Xg) for 2min. The solution obtained by centrifugation is preferably added again to the adsorption column, and the mixture is centrifuged at room temperature for 2min at 12,000rpm (13,400 Xg).
Example 3, comparison of different reagent compositions and concentrations of the kit.
The reagent components and the concentration of the kit are matched with each other, and through the steps of the bacterial genome DNA extraction method, the genome DNA of various gram negative/positive bacteria can be extracted rapidly and efficiently within 1h, and impurity proteins and other metabolic substances in cells can be removed to the maximum extent.
1. Suspension and binding solution.
1) Coli and agrobacterium genomic DNA extraction was tested with suspensions and binding solutions adjusted to the following ingredients. When the components of the suspension and the binding solution are explored, the deproteinized solution used is Buffer PW in the qing biological plant gene DNA extraction kit (TSP 101-50), the rinse solution is the corresponding Wash Buffer, and the eluent is the corresponding TE Buffer. The compositions and concentrations are shown in tables 1 and 2.
TABLE 1
TABLE 2
Table 3 and fig. 1 show the effect of the suspension in combination with the binding liquid. 260 represents the absorbance of the nucleic acid at a wavelength of 260nm of the highest absorbance peak; 280 is the most reactive protein concentration (protein has multiple absorption peaks, 280 is more used); 230 are most reactive to the levels of carbohydrates, salts (e.g., guanidine salts), and other impurities. 260/230 and 260/230 can reflect the purity of nucleic acids to some extent.
For SLS and SDS, the inventors found in experiments that the solution could not be clarified after lysis with SDS, and that the treatment with SLS performed more stably, and that the effect of SLS alone was also superior to that of the combination of SDS and SLS in other experiments. As can be seen from 260/230 of Table 3, the values of the self-assembled B suspensions were 1.27 to 1.876, whereas empirically values around 2.0 indicate a better purity, and values too low may be too high as regards impurity content. It can also be seen from fig. 1 that the strip brightness of suspension a is better overall than that of suspension B. The method shows that the whole extraction product obtained by the treatment of the sarcosyl has higher yield and stable effect.
For the combination solution, 260/280, except for the suspension B+ the value of the root GB is lower, the other values are in a reasonable range; 260/230, the suspension B group is lower overall, the suspension a group is lower, and the combination liquid 2 is lower, and the electrophoresis diagram of fig. 1 is combined, it can be seen that the combination liquids 1 and 3 have better effects, and the sodium acetate solution is selected as a buffer solution component in the combination liquid 2, so that the solution cannot become clear after the pyrolysis and a large amount of flocculent precipitates are formed to plug the column after the ethanol is added; the binding solution 4 is prepared according to the detection result, the binding solution 5 is obtained by adding 1% of AEO-15 on the basis of the binding solution 1, and according to the experimental result, the extraction effect cannot be improved by adding AEO-15.
In addition, it can be seen that when suspensions A and B are used in combination with several different binding fluids, the trend of the change is not consistent, which means that the suspensions and the binding fluids are used cooperatively and cooperatively.
TABLE 3 Table 3
2) On the basis, the effect of extracting the escherichia coli genome DNA is compared by respectively adding different buffers or preparing a binding solution without adding the buffers. Consistent with the results of the previous experiment, the addition of AEO-15 increased the residual amount of RNA in the sample, resulting in higher assay preference and higher purity assay results. No buffer solution is added or Tris-HCl is used, so that the effect of sodium citrate is better. The inventors further compared the use effects of the sodium citrate buffer and Tris-HCl buffer, and found that the use effect of the sodium citrate buffer was better. The use effect of the sodium citrate buffer and the Tris-HCl buffer is further compared by using escherichia coli and agrobacterium, and the effect of using the sodium citrate buffer is better. The compositions and concentrations are shown in table 4. The effect of extracting E.coli/Agrobacterium genomic DNA is shown in Table 5 and FIG. 2 (wherein, (A) represents E.coli and (B) represents Agrobacterium).
TABLE 4 Table 4
TABLE 5
In addition to the above experimental results of discussing the combination of different components, the inventors also carried out experiments on the conditions of the amount of each component, pH value, etc., and determined the optimal ratio of the suspension and the combination liquid as follows: suspension: tris-HCl (pH 8), 50mM; EDTA (pH 8), 10mM; sodium dodecyl sarcosinate (SLS), 1.5%. Binding liquid: sodium citrate (pH 5.0-5.05), 50mM; guHCl,2.5M; urea, 40mM. Wherein the dosage of the suspension is 200 mu L, the dosage of the binding solution is 220 mu L, and the volume dosage ratio of the two is 1:1.1.
2. The components and content of deproteinized liquid.
The reagent components of the earlier stage cracking system have a certain influence on the subsequent purification process of the adsorption column, so that the type and content of salt in the binding solution are matched with the type and content of salt used in the later stage deproteinizing solution, the ideal impurity removing effect can be achieved, and the purity of the product is improved. The E.coli genomic DNA extraction was tested with a protein solution adjusted to the following composition. Further comparative tests were performed below for the better treatments of the above several schemes.
1) A set of deproteinized fluids and results on E.coli. The components and concentrations are shown in Table 6, and the results are shown in Table 7 and FIG. 3 (wherein FIG. (A) is a gel electrophoresis chart of extracted E.coli genomic DNA and FIG. (B) is a gel electrophoresis chart after amplification with different primers using gold-plate MIX). Clearly, the addition of TritonX-100 does not work well.
TABLE 6
TABLE 7
2) The combined effect of adding NaCl, or GuHCl+NaCl, was further investigated. The components and concentrations are shown in Table 8, and the results are shown in Table 9 and FIG. 4 (wherein FIG. (A) is a gel electrophoresis chart of extracted E.coli genomic DNA and FIG. (B) is a gel electrophoresis chart after amplification with different primers using gold-plate MIX). It can be seen that the purity is obviously affected after sodium chloride is added, and in addition, the deproteinized liquid prepared by mixing NaCl and GuHCl has an extraction effect similar to that of the deproteinized liquid prepared by only using GuHCl, but partial impurity residues may have an influence on PCR.
TABLE 8
TABLE 9
On this basis, attempts have been made to add urea to deproteinized solution, which increases product yield, but can significantly affect the product 260/230 ratio and may have an effect on PCR. Experiments in which GuSCN was used instead of GuHCl or in combination with GuHCl have also been tried to find that GuSCN would have residues that would affect subsequent PCR; therefore, guHCl is selected for the deproteinized liquid of the present invention.
3. Buffer concentration and pH effects in deproteinized solutions.
The extraction effect on the genomic DNA of Agrobacterium is shown in Table 10 and FIG. 5.
Table 10
Comparing the concentration of Tris-HCl buffer solution in deproteinized solution and the influence of pH on the extraction effect, the effect is better when the concentration is 30mM; the difference in pH was not significant at 6.8/7.0, but for the purpose of simplifying the post-production, the same pH as that of Tris-HCl buffer in the rinse solution was used at 7.0.
The main components of the deproteinized liquid are determined to be as follows: salts, buffers, alcohols. 2.5M GuHCl is used for manufacturing a high-salt environment, and is matched with the salt concentration in the binding solution, so that protein impurities are effectively removed; tris-HCl is selected as buffer solution, the pH is 7.0, and the concentration is 30mM; in addition, about 55% ethanol was used.
4. Comparison with commercially available products.
The kit of the invention (formula 1, product number TSP 701) was compared with a commercially available similar kit.
Several gram negative and gram positive bacteria were selected and compared with commercially available similar kits using the kit of the present invention (formulation 1) and the results are shown in FIG. 6. It can be seen that the present kit has similar effects to the commercial kits for model bacteria (E.coli, B.subtilis and S.aureus); for some common functional bacteria such as agrobacterium, bacillus thuringiensis, acetic acid bacteria, lactococcus lactis and the like, the effect of the kit is better than that of a commercial product; for other strains, such as pseudomonas aeruginosa, xanthomonas, salmonella typhimurium and enterobacter aerogenes, the kit is obviously superior to the T company, such as staphylococcus epidermidis, streptococcus agalactiae, bacillus licheniformis and rhodococcus, and the kit is obviously superior to the T company and the O company. In general, the kit has good universality and good extraction effect on different bacteria.
Although the invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the invention can be devised without departing from the true spirit and scope of the invention, and the appended claims are intended to be construed to include all such embodiments and equivalent variations. Furthermore, the contents of all references cited herein are hereby incorporated by reference.

Claims (8)

1. A bacterial genome DNA extraction kit comprises a suspension, a binding solution, deproteinized solution, a rinsing solution and an eluent,
the suspension contained 1.5% sarcosyl, 50mM Tris-HCl and 10mM EDTA, pH 8;
the combination liquid comprises 50mM sodium citrate, 2.5M GuHCl and 40mM urea, and the pH value is 5.0-5.05;
the deproteinized solution comprises 30mM Tris-HCl, guHCl of 2.5. 2.5M and ethanol with a final concentration of 55% by volume and a pH of 7;
the rinse solution contained 15mM Tris-HCl and 80% ethanol by volume final concentration, pH 7;
the eluate contained 10 Tris-HCl of mM and 1mM EDTA, pH8.
2. The kit of claim 1, further comprising a DNA adsorption column and proteinase K.
3. The kit of claim 2, wherein the DNA adsorption column is a silicon matrix membrane adsorption column.
4. A method of extracting bacterial genomic DNA using the kit of any one of claims 1-3, comprising:
1) Breaking bacterial cells, degrading proteins and releasing bacterial genomic DNA from the bacterial sample;
2) Adding the binding solution for incubation;
3) Precipitating the DNA with ethanol;
4) Transferring the mixed solution obtained in the last step into an adsorption column for centrifugation, and removing liquid phase;
5) Adding the deproteinized liquid into the adsorption column for centrifugation, and removing liquid phase;
6) Adding the rinsing liquid into the adsorption column for centrifugation, removing liquid phase, and then airing;
7) Eluting DNA from the adsorption column.
5. The method according to claim 4, wherein in 1), the cell walls of gram-negative bacteria are disrupted with the suspension and the cell walls of gram-positive bacteria are disrupted with lysozyme.
6. The method according to claim 4 or 5, wherein in 1), the protein is degraded with proteinase K.
7. The method according to claim 4 or 5, wherein in 2), incubation is performed at 56 ℃ for 10-15 min.
8. The method according to claim 4 or 5, wherein in 6), the centrifugation and the liquid phase removal steps are repeated 1-2 times.
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