CN101413018A - Method for extracting genome DNA - Google Patents
Method for extracting genome DNA Download PDFInfo
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- CN101413018A CN101413018A CNA2008101438535A CN200810143853A CN101413018A CN 101413018 A CN101413018 A CN 101413018A CN A2008101438535 A CNA2008101438535 A CN A2008101438535A CN 200810143853 A CN200810143853 A CN 200810143853A CN 101413018 A CN101413018 A CN 101413018A
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Abstract
The invention discloses a method for extracting genomic DNA. A cell lysate (5-50mmol/L Tris.Cl(pH8.0), 0.2-20mmol/L EDTA(pH8.0),0.1-10 percent SDS) is added into a sample, and a sample cell is lysed; RNase digestion is performed; the sample is cooled, a protein precipitation liquid (1-10mol/L (NH4)2SO4 or 1-10mol/L NH4Ac) is added, and protein is removed through centrifugal precipitation; and a supernatant fluid after the centrifugation is transferred into 100 percent isopropanol, and the DNA is centrifugally precipitated. Compared with the prior art, the method can quickly obtain high-purity high-quality genomic DNA, the whole process only needs 30 minutes, and an obtained DNA fragment is about between 50 and 500kb. And the method has the advantages of simple operation, no need of complicated equipment, low cost, cleanliness, no toxicity, and no harm to users and the environment.
Description
Technical field
The present invention relates to molecular genetics and molecular biology, particularly DNA separation and Extraction and purification process.
Background technology
Molecular biology is very swift and violent in the development of nearly decades, and the research of gene has been spreaded all over medical science, agricultural, environment protection and biological each ambit.The extraction of genomic dna is one of the most frequently used experimental technique of molecular biology, in above-mentioned each field use is arranged all.20 th century later, extracting genomic dna method commonly used is to utilize organic solvent (phenol, chloroform, primary isoamyl alcohol etc.) to make protein denaturation reach the isolating purpose with DNA behind the cell rupture of membranes.These organic solvents have bigger injury to human body skin, respiratory mucosa, eyes etc., and the residual environmental pollution of reagent is bigger; Occurred utilizing the DNA sorbing material to reach protein and the isolating purpose of DNA afterwards again, this method has solved toxicity and pollution problem, but this material cost is higher relatively, operate more loaded down with trivial details, at the biological sample limitation bigger.
Summary of the invention
In order to overcome the problems referred to above, the invention provides a kind of the extraction and the method for purified genomic dna, this method can be at most biological samples, simple to operate, economy, nontoxic pollution-free, high-quality and high-efficiency.
The method of extraction genomic dna of the present invention comprises step: (1) lysed sample cell: press 300ul people's whole blood sample or 10～40mg animal vegetable tissue sample calculation, in sample, add 300ul cell pyrolysis liquid (5～50mmol/L Tris.Cl (pH8.0), 0.2～20mmol/L EDTA (pH8.0), 0.1～10%SDS), the lysed sample cell is blown and beaten mixing back and forth with pipettor; (2) RNase digestion: add RNase solution and the mixing of 4～10mg/ml, 37 ℃ digested 15～60 minutes; (3) albumen precipitation: the sample placement is made its quick cooling on ice, in cooled sample, add 100 μ l albumen precipitation liquid (1～10mol/L (NH4)
2SO
4Or 1～10mol/LNH
4Ac), shake abundant mixing, 13, centrifugal 2 minutes of 000rpm; (4) DNA precipitation: the supernatant liquor after centrifugal is transferred in 100% Virahol, put upside down thorough mixing back and forth, 13, centrifugal 2 minutes of 000rpm, remove supernatant, centrifuge tube is inverted in removes raffinate on the filter paper, add 0.5～1ml, 70% ethanol then, and put upside down centrifuge tube ten back and forth for several times, washing DNA precipitation, 13, centrifugal 1 minute of 000rpm, remove supernatant liquor, centrifuge tube is inverted on the filter paper dry air 10 minutes or vacuum was drained 3～5 minutes; (5) dissolving of DNA: add 50～100 μ l TE or sterilization distilled water, beat mixing, 65 ℃ of water-baths 15 minutes or 37 ℃ of dissolvings are spent the night.Product places 4 ℃ of preservations, preserves for a long time as needs, also can place under-20 ℃ or-80 ℃.
Extraction sample object of the present invention can be whole blood, marrow, culturing cell, animal tissues, plant tissue, gram-positive microorganism, Gram-negative bacteria and yeast.But at different samples, the step that cracking is handled is different.
For culturing cell, collect 1～3 * 10
6Add 300 μ l cell pyrolysis liquids behind the individual cell, blow and beat mixing back and forth with pipettor.
For the mankind and animal tissue specimens, earlier with grinding, method such as shred is handled sample, adds 300 μ l cell pyrolysis liquids in per 10～20mg tissue, blows and beats mixing back and forth with pipettor.Because of protein content is very high, needs to add 55 ℃ of digestion of 1～5 μ l Proteinase K solution (5～40 μ g/ μ l) and spend the night, in the mankind and the animal tissue specimens up to the no macroscopic agglomerate of organizing in cell pyrolysis liquid digestion back.
For blood and bone marrow prepare, need to add earlier 900 μ l erythrocyte cracked liquid (10～500mmol/L NH in per 300 μ l samples
4Cl, 0.1～10mmol/L EDTA, 1～100mmol/L KHCO
3), room temperature was placed 5 minutes, and put upside down mixing frequently back and forth, got limpid until solution becomes.This step purpose is a splitting erythrocyte, and the red corpuscle in peripheral blood and the marrow is acellular nuclear structure, does not contain genomic dna in the cell, so need separate removal with white corpuscle.With the solution after the cracking 13, centrifugal 1 minute of 000rpm, the white corpuscle of visible white precipitation at the bottom of the centrifuge tube, red corpuscle cracking and being distributed in the supernatant liquor.The careful supernatant of removing keeps the supernatant liquor of about 20～30 μ l at last, and white corpuscle is precipitated resuspended with it.In resuspended white corpuscle, add 300 μ l cell pyrolysis liquids, blow and beat mixing back and forth with pipettor.
For the plant tissue sample, in liquid nitrogen, sample is ground earlier with grinding rod.Add 300 μ l cell pyrolysis liquids in per 20～40mg sample, put upside down mixing back and forth after, place 65 ℃ of digestion 60 minutes.
For gram negative bacterium, collect 1ml left and right sides bacterial suspension (about 0.5～2 * 10 with the 1.5ml centrifuge tube earlier
9Individual cell), 13,000rpm collected thalline in centrifugal 15 seconds.Add 300 μ l cell pyrolysis liquids, blow and beat mixing back and forth with pipettor after, place 80 ℃ digestion 5 minutes.
For gram positive bacterium and yeast, collect 1ml left and right sides bacterial suspension (about 1～3 * 10 with the 1.5ml centrifuge tube
8Individual cell), 13,000rpm collected thalline in centrifugal 15 seconds.Add 300 μ l cell suspending liquids (0.1～10mmol/L Nacl, 0.2～50mmol/L Tris.Cl (pH8.0), 0.1～20mmol/L EDTA (pH8.0)), blow and beat mixing back and forth with pipettor.Add 1 μ l N,O-Diacetylmuramidase (20mg/ml) and place 37 ℃ of digestion 30 clocks.Because gram positive bacterium and yeast cell wall are very solid, add the cracking that N,O-Diacetylmuramidase digestion helps cell walls.Digestion product is in 13, centrifugal 1 minute collecting precipitation of 000rpm.In precipitation, add 300 μ l cell pyrolysis liquids, blow and beat mixing back and forth with pipettor after, place 80 ℃ of digestion 5 minutes.
In present method, remove structure agent SDS lysing cell with negatively charged ion, SDS can also suppress the activity of the DNase in the cell or in the environment simultaneously, and DNA is provided stable environment.RNA can remove with the RNA enzymic digestion.When removing protein, use (the NH of high density
4)
2SO
4Solution has replaced traditional organic solution.At last, genomic dna reclaims and is dissolved in the damping fluid that contains the DNA stablizer with ethanol sedimentation.Compared with prior art, present method can obtain the high-purity high-quality genomic dna fast, and whole process can be soon to 30 minutes, the dna fragmentation that obtains is about 50～500Kb, can be used for Southern, PCR, RFLP, various molecular biology experiments such as DNA library construction.And present method is simple to operate, need not complex apparatus, cost is cheap, the more important thing is that cleaning is nontoxic, and is all harmless to user and environment.
Embodiment
Below with embodiment the present invention is specifically addressed, the operation steps described in following all embodiment is all to be operating as example in the 1.5ml centrifuge tube.Extract on a large scale as need, amplify reagent in proportion and sample size gets final product.
Used damping fluid and reagent:
Cell pyrolysis liquid: 5～50mmol/L Tris.Cl (pH8.0), 0.2～20mmol/L EDTA (pH8.0), 0.1～10%SDS;
Erythrocyte cracked liquid: 10～500mmol/L NH
4Cl, 0.1～10mmol/L EDTA, 1～100mmol/L KHCO
3
Cell suspending liquid: 0.1～10mmol/LNacl, 0.2～50mmol/L Tris.Cl (pH8.0), 0.1～20mmol/L EDTA (pH8.0);
Albumen precipitation liquid: 1～10mol/L (NH
4)
2SO
4Or 1～10mol/L NH
4Ac;
Proteinase K solution (5～40 μ g/ μ l);
RNase(4～10mg/ml)；
N,O-Diacetylmuramidase (20mg/ml);
Virahol (100%);
Ethanol (70%).
Embodiment 1: the extraction of culturing cell genomic dna
1) collects 3 * 10
6Individual culturing cell adds 300 μ l cell pyrolysis liquids, blows and beats mixing back and forth with pipettor;
2) RNase solution and the mixing of adding 1.0 μ l 8mg/ml;
3) 37 ℃ digested 30 minutes;
4) sample was placed 1 minute on ice, made its quick cooling;
5) in the refrigerative sample, add 100 μ l albumen precipitation liquid, shake abundant mixing;
6) 13, centrifugal 2 minutes of 000rpm;
7) prepare a new 1.5ml centrifuge tube, add 400 μ l, 100% Virahol;
8) carefully the supernatant liquor after centrifugal is transferred in the Virahol and is put upside down back and forth thorough mixing with pipettor;
9) 13, centrifugal 2 minutes of 000rpm.Manage the visible white agglomerate in the end this moment;
10) remove supernatant, centrifuge tube is inverted in to inhale on the filter paper removes raffinate, add 500 μ l, 70% ethanol then, and put upside down centrifuge tube ten back and forth for several times, washing DNA precipitation;
11) 13, centrifugal 1 minute of 000rpm carefully removes supernatant liquor, centrifuge tube is inverted on the filter paper dry air 10 minutes;
12) add 100 μ l TE, blow and beat mixing with pipettor;
13) 15 minutes dissolving DNAs of 65 ℃ of water-baths;
14) with the DNA sample retention in-20 ℃ of preservations.
Can be with this method from 1～3 * 10
6Extract the genomic dna of 10～30 μ g in the individual culturing cell.
Embodiment 2: the extraction of human peripheral genomic dna
1) add 900 μ l erythrocyte cracked liquids in the human peripheral sample of 300 μ l anti-freezings, room temperature was placed 5 minutes, and put upside down mixing frequently back and forth, got limpid until solution becomes;
2) with the solution after limpid 13, centrifugal 1 minute of 000rpm, visible white precipitate at the bottom of the centrifuge tube; Remove supernatant and discarded with careful suction of 200 μ l pipettors, keep the supernatant liquor of about 20～30 μ l at last, and will precipitate resuspended with it;
3) in resuspended white corpuscle, add 300 μ l cell pyrolysis liquids, blow and beat mixing back and forth with pipettor;
4) all the other steps are with step 2 among the embodiment 1)～step 14).
Can from 300 μ l whole bloods, extract the genomic dna of 5～15 μ g with this method.
Embodiment 3: the extraction of human tissue sample genomic dna
1) get the human tissue sample, earlier with grinding, method such as shred is handled sample;
2) add 300 μ l cell pyrolysis liquids in per 10～20mg tissue, blow and beat mixing back and forth with pipettor;
3) add 1～5 μ l Proteinase K solution in cell pyrolysis liquid digestion back, 55 ℃ of digestion are spent the night, up to the no macroscopic agglomerate of organizing;
4) all the other steps are with step 2 among the embodiment 1)～step 14).
Can from 10～20mg tissue, extract the genomic dna of 10～30 μ g with this method.
Embodiment 4: the extraction of plant tissue sample genomic dna
1), in liquid nitrogen, sample is ground earlier with grinding for the plant tissue sample;
2) add 300 μ l cell pyrolysis liquids in per 20～40mg sample, put upside down mixing back and forth after, place 65 ℃ of digestion 60 minutes
3) all the other steps are with step 2 among the embodiment 1)～step 14).
Can from 20～40mg plant tissue, extract the genomic dna of 5～20 μ g with this method.
Embodiment 5: the extraction of Gram-negative bacteria genomic dna
1) collects 1ml left and right sides Gram-negative bacteria suspension (about 0.5～2 * 10 with the 1.5ml centrifuge tube earlier
9Individual cell), 13,000rpm collected thalline in centrifugal 15 seconds;
2) add 300 μ l cell pyrolysis liquids, blow and beat mixing back and forth with pipettor after, place 80 ℃ of digestion 5 minutes;
3) all the other steps are with step 2 among the embodiment 1)～step 14).
Can from 1ml Gram-negative bacteria suspension, extract the genomic dna of 20 μ g with this way.
Embodiment 6: the extraction of gram-positive microorganism genomic dna
1) collects 1ml left and right sides bacterial suspension (about 1～3 * 10 with the 1.5ml centrifuge tube
8Individual cell), 13,000rpm collected thalline in centrifugal 15 seconds;
2) add 300 μ l cell suspending liquids, blow and beat mixing back and forth with pipettor;
3) add 1 μ l N,O-Diacetylmuramidase and place 37 ℃ of digestion 30 clocks.Because gram positive bacterium and yeast cell wall are very solid, add the cracking that N,O-Diacetylmuramidase digestion helps cell walls;
4) digestion product is in 13, centrifugal 1 minute of 000rpm, collecting precipitation;
5) in precipitation, add 300 μ l cell pyrolysis liquids, blow and beat mixing back and forth with pipettor after, place 80 ℃ of digestion 5 minutes;
6) all the other steps are with step 2 among the embodiment 1)～step 14).
Can from 1ml Gram-positive bacteria suspension, extract the genomic dna of 6～12 μ g with this way.
Claims (7)
1. the extracting method of a genomic dna is characterized in that comprising step:
(1) lysed sample cell: in sample, add cell pyrolysis liquid, blow and beat mixing back and forth with pipettor; Described cell pyrolysis liquid composition is 5～50mmol/L Tris.Cl (pH8.0), 0.2～20mmol/L EDTA (pH8.0), 0.1～10%SDS;
(2) RNase digestion: add RNase solution and the mixing of 4～10mg/mL, 37 ℃ digested 15～60 minutes;
(3) albumen precipitation: sample is cooled off fast, add albumen precipitation liquid, abundant mixing, 13, centrifugal 2 minutes of 000rpm; Described albumen precipitation liquid composition is 1～10mol/L (NH4)
2SO
4Or 1～10mol/LNH
4Ac;
(4) DNA precipitation: the supernatant liquor after centrifugal is transferred in 100% Virahol, put upside down thorough mixing back and forth, 13, centrifugal 2 minutes of 000rpm, remove supernatant, centrifuge tube is inverted in removes raffinate on the filter paper, add 70% ethanol then, and put upside down centrifuge tube ten back and forth for several times, washing DNA precipitation, 13, centrifugal 1 minute of 000rpm, remove supernatant liquor, centrifuge tube is inverted on the filter paper dry air 10 minutes or vacuum was drained 3～5 minutes.
2. the extracting method of genomic dna as claimed in claim 1, it is characterized in that: described sample is a culturing cell; The add-on of described lysate is: per 1～3 * 10
6Add 300 μ l cell pyrolysis liquids in the individual culturing cell.
3. the extracting method of genomic dna as claimed in claim 1 is characterized in that: described sample is the mankind and animal tissues; Described cleavage step (1) is: grind, shred sample earlier, add 300 μ l cell pyrolysis liquids in per 10～20mg tissue, blow and beat mixing back and forth with pipettor; Add 1～5 μ l Proteinase K solution again after the cell pyrolysis liquid digestion, 55 ℃ of digestion are spent the night.
4. the extracting method of genomic dna as claimed in claim 1, it is characterized in that: described sample is blood or marrow; Described cleavage step (1) is: add 900 μ l erythrocyte cracked liquid room temperatures by per 300 μ l samples and placed 5 minutes, and put upside down mixing frequently back and forth, get limpidly until solution becomes, described erythrocyte cracked liquid consists of 10～500mmol/L NH
4Cl, 0.1～10mmol/L EDTA, 1～100mmol/L KHCO
3With the solution after the cracking 13, centrifugal 1 minute of 000rpm removes supernatant, keeps the white corpuscle precipitation, adds 300 μ l cell pyrolysis liquids in the white corpuscle precipitation, blows and beats mixing back and forth with pipettor.
5. the extracting method of genomic dna as claimed in claim 1, it is characterized in that: described sample is a plant tissue; Described cleavage step (1) is: in liquid nitrogen, sample is ground earlier, adds 300 μ l cell pyrolysis liquids by per 20～40mg sample with grinding rod, put upside down mixing back and forth after, place 65 ℃ of digestion 60 minutes.
6. the extracting method of genomic dna as claimed in claim 1, it is characterized in that: described sample is a gram negative bacterium; Described cleavage step (1) is: by 0.5～2 * 10
9Individual somatic cells adds 300 μ l cell pyrolysis liquids, blow and beat mixing back and forth with pipettor after, place 80 ℃ digestion 5 minutes.
7. the extracting method of genomic dna as claimed in claim 1, it is characterized in that: described sample is gram positive bacterium or yeast; Described cleavage step (1) is: by per 1～3 * 10
8Individual somatic cells adds 300 μ l cell suspending liquids, and described cell suspending liquid composition is 0.1～10mmol/L Nacl, 0.2～50mmol/L Tris.Cl (pH8.0), 0.1～20mmol/L EDTA (pH8.0); Blow and beat back and forth with pipettor that to add 1 μ l concentration behind the mixing be the N,O-Diacetylmuramidase of 20mg/ml, place 37 ℃ of digestion 30 clocks; Digestion product is in 13, centrifugal 1 minute of 000rpm, collecting precipitation; In precipitation, add 300 μ l cell pyrolysis liquids, blow and beat mixing back and forth with pipettor after, place 80 ℃ of digestion 5 minutes.
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| CN112945667A (en) * | 2021-02-01 | 2021-06-11 | 海南师范大学 | Preparation method of blood single cell suspension for removing nucleated red blood cells |
| CN112945667B (en) * | 2021-02-01 | 2024-03-26 | 海南师范大学 | Preparation method of blood single-cell suspension for removing nucleated red blood cells |
| CN113265397A (en) * | 2021-06-01 | 2021-08-17 | 上海捷瑞生物工程有限公司 | Cell lysate, kit and method for yeast genome extraction |
| CN114752655A (en) * | 2022-05-17 | 2022-07-15 | 安徽荃银高科种业股份有限公司 | DNA extraction method suitable for molecular marker-assisted breeding |
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