CN103911366A - Genome DNA extraction method and its kit - Google Patents
Genome DNA extraction method and its kit Download PDFInfo
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Abstract
The invention discloses a genome DNA extraction method and a kit. The kit comprises a grinder, a solution A(lysate), a solution B(DNA combination liquid), a solution C(DNA washing liquid), a solution D(DNA eluate) and a purifying column. The provided kit has the advantages that cost is low, the reagent component is simple, the operation flow is little, and the kit is suitable for extraction of microbe genome and is also suitable for extraction of animal tissue genome and plant tissue genome. The time consuming of a process for extracting the genome is short (in 30 minutes), the quality of the extracted genome is high, the size of the genome strip is 23kb, the genome amount can be kept in a scope of 1mumg-30mumg in a stable mode; a toxic reagent phenol and chloroform are not used in the extraction process, and various molecular biology experiments for SouthernBlot, PCR, RFLP, DNA library construction can be better satisfied.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to genome DNA extracting reagent kit and extracting method.
Background technology
Since pcr amplification technological invention, the research of biomolecular science is advanced by leaps and bounds.Own through spreading all over medical treatment & health, agriculture production, environment protection and biological each ambit to the research of gene.The extraction of genomic dna is the prerequisite of molecular biology research, widespread use in above-mentioned each field.Extracting the conventional method of genomic dna is to use physical method broken wall, for example liquid nitrogen grinding and heat; Or enzymolysis, for example N,O-Diacetylmuramidase enzymolysis; Or nonionogenic tenside processing example as, CTAB method (Walker WH etc., 1991) Benzyl chloride method and SDS method (Lin Fucheng and Wang Hongkai, 2010), cell wall breaking makes protein denaturation reach the object separating with DNA with the mixing solutions that utilizes organic solvent use phenol, chloroform, primary isoamyl alcohol after rupture of membranes.Although these methods can extract high-quality genomic dna, but these organic solvents in leaching process have larger injury to human body skin, respiratory mucosa, eyes etc., and extract genome often have organic solvent residual, make experimental result below unstable; Occurred again afterwards utilizing DNA sorbing material to reach the object that protein separates with DNA, and simultaneously also developed a lot of test kits and extract the genome of differing materials, although the genome extracting is of high quality, cost is higher, versatility is not strong.No matter but be to extract genome with the extracting of the organic solvent such as phenol, chloroform or with DNA extraction test kit, the pre-treatment of sample is a process loaded down with trivial details and consuming time all the time.Gram positive bacterium and yeast cell wall are very solid, all can add N,O-Diacetylmuramidase peptic cell wall in common extraction genome method.For human and animal tissue, need to add Proteinase K to spend the night to process to make to organize more and disperse.For plant tissue and filamentous fungus mycelia or fungal spore, need to use liquid nitrogen grinding broken wall.
Summary of the invention
In order to overcome the problems referred to above, the invention provides a kind of rapid gene group DNA extraction test kit and preparation method thereof, the method can be for most biological samples, simple to operate, economy, nontoxic pollution-free, high-quality and high-efficiency.
A kind of genome DNA extracting reagent kit, comprises solution A (lysate), solution B (DNA is in conjunction with liquid), solution C (DNA washings) and solution D (DNA elutriant);
Solution A (lysate): 10mM-50mM Tris-HCl pH8.0,10mM-50mM EDTA, 1%-10%(w/v) SDS, 2M-4M NaCl, 0.5%-2%(v/v) beta-mercaptoethanol;
Solution B (DNA is in conjunction with liquid): the Guanidinium hydrochloride of 2.5-6M or guanidinium isothiocyanate, 20%-40%(v/v) Virahol or dehydrated alcohol, 5mM-20mM EDTA, pH4.5-6.5;
Solution C (DNA washings): dehydrated alcohol 70%-80%(v/v), pH4.5-6.5;
Solution D (DNA elutriant): the RNA enzyme aqueous solution of 5-50 μ g/mL, pH is transferred to 7.0-8.5 with 1M NaOH solution.
Also comprise mill: this mill can be electronic tissue grinder or small-sized its rotating speed of electrichand drill 800-12000rpm, and being furnished with length is 40-80mm, and pestle is ground in the taper of diameter 3-7mm.
The screening formulation of mentioned reagent box
Solution A: 50mM Tris-HCl, 50mM EDTA, 3%SDS, 2M NaCl, 1% beta-mercaptoethanol, pH8.0;
Acetic acid-sodium acetate buffer of the 0.2M of the Guanidinium hydrochloride of solution B: 4M, 20% Virahol, 10mM EDTA, pH5.0;
Solution C: acetic acid-sodium acetate buffer of 70% dehydrated alcohol, the 0.2M of pH5.0
Solution D: the RNA enzyme aqueous solution of 20 μ g/mL, pH is transferred to 8.0 with 1M NaOH solution.Mill: rotating speed 8000rpm, to be furnished with length be 70mm, pestle is ground in the taper of diameter 5mm.
Cardinal principle of the present invention: the shearing force that mill produces under high-speed condition can not only be by the cell walls fragmentation of filamentous fungus, the rubbing effect of simultaneous grinding pestle and centrifugal tube wall and produce higher temperature (50 DEG C-70 DEG C), is conducive to SDS lysing cell.Under the interaction of mill, high temperature and SDS, the genomic dna of filamentous fungus discharges.The solubleness maximum of DNA in 2M NaCl solution.EDTA is the activity that metal chelator can suppress DNA enzyme, prevents the degraded of DNA.Beta-mercaptoethanol is antioxidant, effectively prevents that phenol is oxidized to quinone, avoids brown stain, and phenol is easily removed.DNA provides the environment of a low pH value of high salt in conjunction with liquid, under this condition, DNA can with silicagel column Reversible binding.Washings is that 70% ethanol can be to silicagel column desalination.DNA, in the low salts solution of pH7.0-8.5, has maximum elution efficiency.Elutriant is the alkaline aqueous solution that contains RNA enzyme, DNA on silicagel column can be eluted.
Another aspect of the present invention is to use this test kit to extract the method for genomic dna, comprises the following steps:
1, the pre-treatment of sample
(1) get centrifuge tube at the bottom of the cone of 1.5mL, add 0.1mg-100mg tissue sample or 10
6-10
9cell sample;
(2) to the solution A that adds 50 μ L-100 μ L in (1);
(3) start mill ground sample 30s-5min;
2, the column purification excessively of sample
(4) in ground mycelia, add solution A to make sample final volume in 150 μ L left and right;
(5) vortex oscillation device concussion 30s disperses thalline as far as possible;
(6) centrifuge speed 10000g-12000g, centrifugal 5min;
(7) carefully get in the 1.5mL centrifuge tube that supernatant 100 μ L to are new, add the solution B of 300 μ L-600 μ L, turn upside down and mix;
(8) mixed solution of (7) is added in purification column to 10000g-12000g, centrifugal 30s-1min;
(9) abandon the liquid in collection tube, to the solution B that adds 300 μ L-600 μ L in purification column, 10000g-12000g, centrifugal 30s-1min;
(10) abandon the liquid in collection tube, to the solution C that adds 500 μ L-700 μ L in purification column, 10000g-12000g, centrifugal 30s-1min;
(11) repeat (10);
(12) abandon the liquid in collection tube, 10000g-12000g, centrifugal 2-5min;
(13) purification column of (12) is transferred in a new 1.5mL centrifuge tube, added the solution D of 30-50 μ L, place 5min for 37 DEG C;
(14) 10000g-12000g, centrifugal 1-2min;
(15) preserve the genomic dna solution extracting for-20 DEG C.
The object that the present invention extracts sample can be that culturing cell (zooblast or human cell) or bacterium (gram negative bacterium and gram positive bacterium) or yeast etc. are unicellular, the mankind and animal tissues or plant tissue or macro fungi tissue, blood or marrow, filamentous fungus (aspergillus oryzae, aspergillus niger etc.), fungal spore (aspergillus oryzae spore, aspergillus niger spore, Ganoderma spore etc.).But the sample difference of processing, the step of cracking processing is different.
A, be that culturing cell (zooblast or human cell) or bacterium (gram negative bacterium and gram positive bacterium) or yeast etc. are unicellular for sample: described sample pretreatment step is: get 1mL concentration>=1.0 × 10
6the cell of/mL, centrifugal 5min collecting cell in the whizzer of rotating speed>=5000rpm, adds the lysate of 50 μ L-100 μ L, and grinds 1-2min with mill.
B, be the mankind and animal tissues or plant tissue or macro fungi tissue for sample, for example, mushroom, auricularia auriculajudae etc.; Described pre-treatment step is: sample is shredded or ground, transfer in the centrifuge tube of 1.5mL, sample size is controlled at the lysate that 0.1mg-100mg adds 50 μ L-100 μ L, and grinds 3-5min with mill.
C, be blood or marrow for sample; Described pre-treatment step is: get 1mL blood, centrifugal 5min collecting cell in the whizzer of rotating speed >=5000rpm, adds the lysate of 50 μ L-100 μ L, and grinds 1-2min with mill.
D, be filamentous fungus, such as aspergillus oryzae, aspergillus niger etc. for sample.Can be with toothpick or pipettor gun head picking for being grown in mycelia on solid medium; Can or filter collection mycelia with centrifugal 10min in the whizzer of rotating speed >=10000rpm for being grown in mycelia in liquid nutrient medium.Mycelia amount adds the lysate of 50 μ L-100 μ L at 0.1mg-100mg, and grinds 1-2min with mill.
E, be fungal spore for sample, such as aspergillus oryzae spore, aspergillus niger spore, Ganoderma spore etc.Described pre-treatment step is: can collect spore or use the mode of filtering collect spore with centrifugal 10min in the whizzer of rotating speed >=10000rpm, add the lysate of 50 μ L-100 μ L, and grind 3-5min with mill.
Compared with prior art, tool has the following advantages:
Present method is mainly to adopt mechanical mill smudge cells and in conjunction with SDS lysing cell, do not need to use various lyase, for example N,O-Diacetylmuramidase, Proteinase K, and follow-up genome column purification reagent composition is simple, and operating process is few; This test kit is applied widely, be not only applicable to the extraction of microbial genome but also be applicable to animal tissues and the genomic extraction of plant tissue, can obtain fast purity height and the high genomic dna of quality simultaneously, whole process control is in 30 minutes, the DNA fragmentation obtaining is about 23Kb, genomic amount can be stablized and remains within the scope of 1 μ g-30 μ g, can not use poisonous reagent phenol, chloroform, be well positioned to meet follow-up experiment, can be used for Southern, PCR, RFLP, the various molecular biology experiments such as DNA enzyme is cut, DNA library construction.And present method is simple to operate, it is cheap to spend, the more important thing is clean nontoxic, all harmless to user and environment.
Brief description of the drawings
Fig. 1 is for using this test kit to extract the genomic electrophorogram of aspergillus oryzae RIB40.
Fig. 2 produces the genomic electrophorogram of spore aspergillus niger CBS513.88 for using this test kit to extract.
Fig. 3 is for using this test kit to extract without the genomic electrophorogram of spore aspergillus niger SH-2.
Fig. 4 is for using this test kit to extract the genomic electrophorogram of aspergillus oryzae RIB40 conidium.
Fig. 5 is for using this test kit to extract the genomic electrophorogram of edible fungus black fungus.
Fig. 6 is for using this test kit to extract the genomic electrophorogram of edible fungus mushroom.
Fig. 7 is for using this test kit to extract the genomic electrophorogram of edible fungus flat mushroom.
Fig. 8 is for using this test kit to extract the genomic electrophorogram of edible fungus tea tree mushroom.
Fig. 9 is for using this test kit to extract the genomic electrophorogram of yeast GIM2.8.
Figure 10 is for using this test kit to extract the genomic electrophorogram of subtilis WB800.
Figure 11 is for using this test kit to extract the genomic electrophorogram of intestinal bacteria JM110.
Figure 12 is for using this test kit to extract the genomic electrophorogram of culturing cell.
Figure 13 is for using this test kit to extract the genomic electrophorogram of plant tissue leaf.
Figure 14 uses this test kit to extract the electrophorogram of animal tissues's porky genome.
Figure 15 uses this test kit to extract the electrophorogram of animal tissues's fish viscera tissue gene group.
The Maker that digital M representative in accompanying drawing is used is λ-Hind III digest Maker of precious biotechnology (Dalian) company limited of use, and stripe size is respectively 23130bp, 9416bp, 6557bp, 4361bp, 2322bp, 2027bp, 564bp, 125bp from top to bottom.Numeral 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 represents respectively the genome of different samples in corresponding diagram.
Embodiment
By embodiment, the present invention is specifically addressed below, the operation steps described in following all embodiment is all operating in centrifuge tube at the bottom of the cone of 1.5mL.Following instance is to further illustrate of the present invention, should not do limitation of the present invention.In this example, material aspergillus oryzae RIB40, aspergillus niger CBS513.88, aspergillus niger SH-2, intestinal bacteria JM110, subtilis WB800, Candida utilis bacterium GIM2.8 used is that preserve in this laboratory, also can adopt commercially available prod; People's culturing cell is that South China Science & Engineering University's bio-science and doctor Ding Xiaoru of engineering college are so kind as to give; Black fungus, mushroom, flat mushroom, tea tree mushroom, pork, fish viscera are organized and are all purchased from food market, Sui Shi village of Guangzhou university city; Leaf tissue is plucked in South China Science & Engineering University.
Embodiment 1
The genomic extracting method of aspergillus oryzae mycelia
1, following formula is made aspergillus oryzae mycelia genome and is extracted test kit:
Mill: rotating speed 8000rpm, to be furnished with length be 70mm, pestle is ground in the taper of diameter 5mm;
Solution A: 50mM Tris-HCl, 50mM EDTA, 3%SDS, 2M NaCl, 1% mercaptoethanol, pH8.0;
Acetic acid-sodium acetate buffer of the 0.2M of the Guanidinium hydrochloride of solution B: 4M, 20% Virahol, 10mM EDTA, pH5.0;
Solution C: acetic acid-sodium acetate buffer of 70% dehydrated alcohol, the 0.2M of pH5.0;
Solution D: the RNA enzyme aqueous solution of 20 μ g/mL, pH is transferred to 8.0 with 1M NaOH solution.
DNA purification column: silicagel column.
Wherein, mill is purchased from Tian Gen biochemical technology company limited.
2, complete the genomic extraction of aspergillus oryzae according to following steps:
The pre-treatment of a, sample
(1) get centrifuge tube at the bottom of the cone of 1.5mL, add the aspergillus oryzae RIB40 thalline of 10mg;
(2) to the solution A that adds 100 μ L in the centrifuge tube of (1);
(3) grind thalline 2min with mill;
B, sample are crossed column purification
(4) in ground mycelia, add solution A to make final volume in 150 μ L left and right;
(5) at vortex oscillation device concussion 30s, thalline is disperseed as far as possible;
(6) centrifuge speed 12000g, centrifugal 5min;
(7) carefully get in the 1.5mL centrifuge tube that supernatant 100 μ L to are new, add the solution B of 300 μ L, turn upside down and mix;
(8) mixed solution of (7) is added in purification column to 12000g, centrifugal 1min;
(9) outwell the liquid in collection tube, to the solution B that adds 500 μ L in purification column, 12000g, centrifugal 1min;
(10) outwell the liquid in collection tube, to the solution C that adds 700 μ L in purification column, 12000g, centrifugal 1min;
(11) repeat (10);
(12) outwell the liquid in collection tube, 12000g, centrifugal 3min;
(13) purification column of (12) is added in a new 1.5mL centrifuge tube, add the solution D of 30 μ L, place 5min for 37 DEG C;
(14) 12000g, centrifugal 2min;
(15) preserve the genomic dna solution extracting for-20 DEG C.
C, the genome that uses the method to extract can extract approximately 1.5 μ g from 10mg thalline, and the genome of extraction as shown in Figure 1.
Embodiment 2
Produce the genomic extraction of spore aspergillus niger CBS513.88 mycelia
Extraction Methods of Genome is with the step in embodiment 1, and the genome that uses the method to extract can extract approximately 1.5 μ g from 10mg thalline, and the genome of extraction as shown in Figure 2.
Embodiment 3
Without the genomic extraction of spore aspergillus niger SH-2 mycelia
In genome leaching process, step method is with the step in embodiment 1, and the genome that uses the method to extract can extract approximately 2 μ g from 10mg thalline, and the genome of extraction as shown in Figure 3.
Embodiment 4
The genomic extraction of aspergillus oryzae conidium
Extraction Methods of Genome, with embodiment 1, just changes step (3) in embodiment 1 into mill grinding spore 5min, and the genome that uses the method to extract can be from 1 × 10
8in the spore of amount, extract approximately 1.5 μ g, the genome of extraction as shown in Figure 4.
Embodiment 5
The genomic extraction of edible fungus black fungus
Extraction Methods of Genome, with embodiment 1, just changes step (3) in embodiment 1 into mill grinding thalline 5min, and the genome that uses the method to extract can extract approximately 2 μ g in the black fungus of 10mg, and the genome of extraction as shown in Figure 5.
Embodiment 6
The genomic extraction of edible fungus mushroom
Extraction Methods of Genome, with embodiment 1, just changes step (3) in embodiment 1 into mill grinding thalline 5min, and the genome that uses the method to extract can extract approximately 2 μ g in the mushroom of 10mg, and the genome of extraction as shown in Figure 6.
Embodiment 7
The genomic extraction of edible fungus flat mushroom
Extraction Methods of Genome, with embodiment 1, just changes step (3) in embodiment 1 into mill grinding thalline 5min, and the genome that uses the method to extract can extract approximately 2 μ g in the flat mushroom of 10mg, and the genome of extraction as shown in Figure 7.
Embodiment 8
The genomic extraction of edible fungus tea tree mushroom
Extraction Methods of Genome, with embodiment 1, just changes step (3) in embodiment 1 into mill grinding thalline 5min, and the genome that uses the method to extract can extract approximately 2 μ g in the tea tree mushroom of 10mg, and the genome of extraction as shown in Figure 8.
Embodiment 9
The genomic extraction of Candida utilis bacterium GIM2.8
Genome extracts with the method described in embodiment 1, and the genome that uses the method to extract can be from 1 × 10
9in the yeast cell of amount, extract approximately 3 μ g, the genome of extraction as shown in Figure 9.
Embodiment 10
The genomic extraction of gram positive bacterium subtilis WB800
Genome extracts with the method described in embodiment 1, and the genome that uses the method to extract can be from 1 × 10
9in the withered grass cell of amount, extract approximately 5 μ g, the genome of extraction as shown in figure 10.
Embodiment 11
The genomic extraction of gram negative bacterium intestinal bacteria bacillus JM110
Genome extracts with the method described in embodiment 1, and the genome that uses the method to extract can be from 1 × 10
9in the colorectal cell of amount, extract approximately 5 μ g, the genome of extraction as shown in figure 11.
Embodiment 12
The genomic extraction of people's culturing cell
Genome extracts with the method described in embodiment 1, and the genome that uses the method to extract can be from 1 × 10
9in people's cell of amount, extract approximately 6 μ g, the genome of extraction as shown in figure 12.
Embodiment 13
The genomic extraction of plant tissue plant leaf
Extraction Methods of Genome, with embodiment 1, just changes step (3) in embodiment 1 into mill grinding plant leaf 5min, and the genome that uses the method to extract can extract approximately 2 μ g in the plant leaf of 10mg, and the genome of extraction as shown in figure 13.
Embodiment 14
The extraction of animal tissues's porky genome
Extraction Methods of Genome, with embodiment 1, just changes step (3) in embodiment 1 into mill grinding pork 5min, and the genome that uses the method to extract can extract approximately 1.5 μ g in the pork of 10mg, and the genome of extraction as shown in figure 14.
Embodiment 15
The extraction of animal tissues's fish viscera tissue gene group
Extraction Methods of Genome, with embodiment 1, just changes step (3) in embodiment 1 into mill grinding fish viscera 5min, and the genome that uses the method to extract can extract approximately 2 μ g in the fish viscera of 10mg, and the genome of extraction as shown in figure 15.
Claims (9)
1. a genome DNA extracting reagent kit, is characterized in that, comprises solution A, solution B, solution C, solution D and purification column;
Solution A: 10mM-50mM Tris-HCl, 10mM-50mM EDTA, 1%-10%w/v SDS, 2M-4M NaCl, 0.5%-2%v/v beta-mercaptoethanol, pH7.5-8.5;
The Guanidinium hydrochloride of solution B: 2.5-6M or guanidinium isothiocyanate, 20%-50%v/v Virahol or dehydrated alcohol, 5mM-20mM EDTA, pH4.5-6.5;
The dehydrated alcohol of solution C: 70%-80%v/v, pH4.5-6.5;
Solution D: the RNA enzyme aqueous solution of 5-50 μ g/mL, with 1M NaOH solution tune pH to 7.0-8.5.
2. test kit according to claim 1, is characterized in that, also comprises mill, and this mill can be electronic tissue grinder or small-sized its rotating speed of electrichand drill 800-12000rpm, and being furnished with length is 40-80mm, and pestle is ground in the taper of diameter 3-7mm.
3. test kit according to claim 2, is characterized in that, the rotating speed 1500-8000rpm of described mill is also furnished with length 70mm, and pestle is ground in the taper of diameter 5mm.
4. according to the test kit described in claim 1 or 2 or 3, it is characterized in that,
Solution A: 50mM Tris-HCl, 50mM EDTA, 3%SDS, 2M NaCl, 1% beta-mercaptoethanol, pH8.0;
Acetic acid-sodium acetate buffer of the 0.2M of the Guanidinium hydrochloride of solution B: 4M, 20% Virahol, 10mM EDTA, pH5.0;
Solution C: acetic acid-sodium acetate buffer of 70% dehydrated alcohol, the 0.2M of pH5.0;
Solution D: the RNA enzyme aqueous solution of 20 μ g/mL, pH is transferred to 8.0 with 1mol/L NaOH solution.
5. a genome DNA extracting method, is characterized in that, utilizes the test kit described in claim 1～4 any one to extract, and specifically comprises following steps:
(1) get centrifuge tube at the bottom of the cone of 1.5mL, add 0.1mg-100mg tissue samples or 10
6-10
9cell sample mixes with the solution A of 50 μ L-100 μ L; Starting mill and grind sample 30s-5min, is 150 μ L to adding solution A to make sample final volume in ground sample;
(2) vortex oscillation device concussion 30s disperses thalline as far as possible; Centrifugal rotational speed 10000g-12000g, time 5min; Get in supernatant 100 μ L to centrifuge tubes, add the solution B of 300 μ L-600 μ L, turn upside down and mix; Again mixed solution is added in purification column to 10000g-12000g, centrifugal 30s-1min; Abandon the liquid in collection tube, to the solution B that adds 300 μ L-600 μ L in purification column, 10000g-12000g, centrifugal 30s-1min;
(3) abandon the liquid in collection tube, to the solution C that adds 500 μ L-700 μ L in purification column, 10000g-12000g, centrifugal 30s-1min; Abandon the liquid in collection tube, to the solution C that adds 500 μ L-700 μ L in purification column, 10000g-12000g, centrifugal 30s-1min; Abandon the liquid in collection tube, 10000g-12000g, centrifugal 2-5min;
(4) purification column is transferred in another centrifuge tube, added the solution D of 30-50 μ L, place 5min for 37 DEG C; 10000g-12000g, centrifugal 1-2min; Obtain the genomic dna solution extracting.
6. method according to claim 5, is characterized in that, described cell sample is mammalian cell, bacterium, yeast, blood or marrow; Described cell sample is also through following pre-treatment: by cell sample centrifugal 5min collecting cell in the whizzer of rotating speed >=5000rpm.
7. method according to claim 5, is characterized in that, described tissue samples is animal vegetable tissue or macro fungi tissue; This tissue samples is the pre-treatment through shredding or grinding also.
8. method as claimed in claim 5, is characterized in that, described tissue samples is filamentous fungus, for the mycelia toothpick or the pipettor gun head picking that are grown on solid medium; For being grown in mycelia centrifugal 10min in the whizzer of rotating speed >=10000rpm or filtration collection mycelia in liquid nutrient medium.
9. method as claimed in claim 5, is characterized in that, described tissue samples is fungal spore suspension, and in the whizzer with rotating speed >=10000rpm, centrifugal 10min collects spore or uses the mode of filtering to collect spore.
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| CN118326063A (en) * | 2024-03-25 | 2024-07-12 | 中国热带农业科学院热带作物品种资源研究所 | Okra germplasm resource genetic diversity analysis method |
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