CN101338311A - Method for extracting total DNA from milk - Google Patents
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- CN101338311A CN101338311A CNA2008101158229A CN200810115822A CN101338311A CN 101338311 A CN101338311 A CN 101338311A CN A2008101158229 A CNA2008101158229 A CN A2008101158229A CN 200810115822 A CN200810115822 A CN 200810115822A CN 101338311 A CN101338311 A CN 101338311A
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Abstract
The invention belongs to the technology researching field of cow molecular, in particular to the technical field of preparing the total DNA of the cow. The invention discloses a method for extracting the total DNA from the cow. Because the cow belongs to an economic animal, in order to avoid the stress caused by blood sampling and collecting the tissue of a living cow as well as overcome the problem of containing higher protein pollution of the extracted total DNA of a milk sample, the invention proposes a more convenient source to obtain the DNA, researches out a key technology from purifying the somatocyte of the milk sample to extracting the DNA with high quality, prepares the reagent for purifying the somatocyte of the milk sample and a cracking mixture for extracting the DNA by test, then uses alcohol with different concentration grads to wash, then uses a phenol method to extract and remove the protein and other matters like amylase, finally uses absolute ethyl alcohol to wash and dissolves into TE after being dried. The invention is simple and convenient to sample; the quality of the sample of the total DNA that is extracted is higher; the method is economic, stable and convenient; the invention provides a novel method for the application of the cow molecular.
Description
Technical field
The present invention relates to the milk cow technical field of molecular biology.Be specifically related to the method for purifying somatocyte and the total DNA of extracting from milk sample.
Background technology
Obtaining genomic dna is the basis of carrying out subsequent molecular, seeking suitable DNA extraction method is to carry out first problem that most of molecular biology experiment faces, use at present method acquisition milk cow DNA to be the major issue that the investigator faces, especially show the animal welfare aspect, major cause is owing to take blood sample can cause stress reaction, reduces milk cow production performance.The current source from the individual sample that obtains genomic dna of milk cow mainly contains: blood, muscle tissue, organs and tissues, seminal fluid etc., and conventional extracting method is used in this several samples source, and the effect of extraction is better.Utilize milk to extract the source at present, have the deficient problem of technology as high quality DNA.The method that current utilization milk sample extracts DNA as the source mainly contains salting-out process (Tian Yu, the foundation of DNA isolation method from milk; The dairy industry science; 2006,3:112-113.F.d ' Angelo, A.Santillo, A.Sevi, and M.Albenzio (2007) adopts salting-out process to extract the total DNA of goat milk (A Simple Salting-Out Methodfor DNA Extraction from Milk Somatic Cells:Investigation into the Goat CSN1S1 gene .J.DairySci.90:3550-3552) .) and SDS/ phenol method (E.LIPKIN, ANNE SHALO M, H.KHATIB, M.SOLLER, and A.FRIEDMANN (1993) .Milk as a Source of Deoxyribonucleic Acid and as a Substrate for the PolymeraseChain Reaction.J Dairy Sci 76:2025-2032), all be to obtain somatocyte earlier, and then digest extracting with reagent corresponding.Therefore with regard to present technology, the milk sample DNA that obtains is relatively low in purity and concentration, needs more to improve one's methods, and has reached the acquisition sample DNA sample of suckling preferably.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, set up a kind of from milk the method for the total DNA of extracting, with reach fast, economic, effect easily, be applicable to the molecular biological technological operation of milk cow.
The present invention is achieved through the following technical solutions:
A kind of method of extracting total DNA from milk is characterized in that following steps:
1, a kind of method of extracting total DNA from milk is characterized in that following steps:
1) prepare before the sampling: clean mammilla of milk cattle with the tincture of iodine, dry the Supramammillary tincture of iodine with clean warm water cloth, the preceding 2-3 that abandons extruding obtains the milk sample to milk with milking apparatus or hand milking's method afterwards;
2) sampling: add 1ml somatocyte stationary liquid and 20% potassium bichromate 0.5ml in advance as sanitas in centrifuge tube, pack the milk sample of taking into centrifuge tube to 50ml, in 2500 rev/mins, 4 ℃ centrifugal 30 minutes;
3) discard step 2) the upper strata butterfat and the middle layer emulsion of centrifuge tube, keep its bottom settlings, add 1ml ditalimfos acid buffer to the bottom, bottom settlings beaten suspend and be transferred in the centrifuge tube of 1.5ml, at 3500 rev/mins, normal temperature (20-25 ℃) centrifugal 10 minutes down discards supernatant liquid, keeps bottom settlings;
4) in the centrifuge tube of step 3), add the phosphoric acid buffer 1ml of sterilization, the precipitation that suspends, with the vibrator vibration till precipitation suspends fully, then at 1000 rev/mins, under the normal temperature centrifugal 10 minutes;
5) after centrifugal supernatant is discarded, add emulsifying agent 150 μ l, add the phosphoric acid buffer 1350 μ l of sterilization again, suspend fully with vibrator vibration to precipitation, the butterfat of sloughing in 5-10 minute around the somatocyte is handled in 40 ℃ of waters bath with thermostatic control;
6) after the water-bath, at 3500 rev/mins, under the normal temperature centrifugal 10 minutes, abandon supernatant after centrifugal, add the phosphoric acid buffer 1ml precipitation that suspends, mixed solution is transferred in the centrifuge tube of 1.5ml, made the somatocyte precipitation in centrifugal 10 minutes in 12000 rev/mins, with the somatocyte that obtains put-20 ℃ frozen, perhaps directly described somatocyte is carried out DNA extraction;
7) have adding cracking mixed liquor I 600 μ l and cracking mixed liquor I I 150 μ l in the somatic 1.5ml centrifuge tube to step 6), vibration makes its bottom settlings to suspending fully, places 56 ℃ of water-bath digested overnight, obtains digestion product;
8) digestion product to step 7) adds isopyknic dehydrated alcohol, and 12000 rev/mins of centrifugations discard top ethanol, 95%, 75%, 70% ethanol that adds 1ml then respectively carries out the gradient washing, discard the ethanol on upper strata respectively, ethanol evaporation is 5 minutes at normal temperatures, obtains the DNA crude product;
9) the DNA crude product that step 8) is obtained adds 500 μ l deionization aqua sterilisas makes its dissolution precipitation, and the pH that adds two volumes again is that 8.0 the saturated phenol solution of Tris carries out extracting, puts upside down back and forth 10 minutes, 12000 rev/mins, under the normal temperature centrifugal 10 minutes, obtains supernatant liquor;
10) supernatant liquor of step 9) being transferred in another 1.5ml centrifuge tube, put upside down back and forth 10 minutes, is the phenol of 25: 24: 1 adding two volumes: chloroform by volume: primary isoamyl alcohol, and at 12000 rev/mins, extracting 10 minutes obtains supernatant liquor;
11) supernatant liquor of step 10) being transferred in another 1.5ml centrifuge tube, is 24: 1 adding two volumes chloroforms by volume: primary isoamyl alcohol, put upside down back and forth 10 minutes, and 12000 rev/mins, extracting 10 minutes obtains supernatant liquor;
12) supernatant liquor of step 11) is transferred in another 1.5ml centrifuge tube, added the 3M/L of 1/10 volume, pH is the dehydrated alcohol of the precooling of 5.2 NaAc and 2 times of volumes, rocks centrifuge tube back and forth, places 30 minutes in-20 ℃; In 12000 rev/mins, 25 ℃ were descended centrifugal 10 minutes, carefully outwelled the upper strata dehydrated alcohol, obtained the DNA white precipitate again;
13) the ethanol 1ml of adding 70%, washing step 12) described DNA white precipitate, in centrifugal 10 minutes of following 12000 rev/mins of normal temperature, carefully discard ethanol, the residual ethanol of volatilizing at normal temperatures, 50-100 μ l three (methylol) aminomethane-ethylenediamine tetraacetic acid (EDTA) (Tris-EDTA/TE) that the adds dissolving DNA that spends the night obtains the pure product of DNA;
Wherein,
Step 2) described somatocyte stationary liquid is formulated as follows:
35-40% formaldehyde 9.4ml is dissolved in the 100ml distilled water surely;
The described emulsifying agent of step 5) is formulated as follows:
In the 1L emulsifying agent: 90% Triton X-100 (triton-x 100) 20ml, 95% ethanol 125ml, 0.9g/l NaCl solution 855ml;
The described cracking mixed liquor I of step 7) is formulated as follows:
Mixed liquor I: NaCl 1M/L, Tris-Cl 0.5M/L, EDTA-Na 0.5M/L transfers pH to 7.5-8.0;
The described cracking mixed liquor I of step 7) I is formulated as follows:
20% sodium lauryl sulphate, 40 μ l, the Proteinase K 20 μ l of 20mg/ml, emulsifying agent 90 μ l;
The described Tris-EDTA/TE solution of step 13) is formulated as follows:
Get pH respectively and be 8.0 1M/L Tris-Cl 2ml and 0.5M/L EDTA 0.4ml, its two kinds of solution are mixed, adding distil water is settled to 200ml, transfers pH to 8.0.
Step 3), 4), 5) and 6) described phosphoric acid buffer is formulated as follows:
Take by weighing NaCl 8g respectively, KCL 0.2g, Na
2HPO
41.44g, KH
2PO
40.24g, various substance dissolves in 800ml distilled water, are transferred pH to 7.4, adding distil water is settled to 1L.
2, in " summary of the invention " 1 used milk sample be behind the adding preservative agent 24 hours with interior milk sample.
The invention has the beneficial effects as follows:
The present invention is mainly reflected in following aspect to the contribution of prior art:
The present invention adds somatocyte stationary liquid, sanitas and emulsifying agent in the somatocyte purifying, the somatocyte stationary liquid makes the somatocyte in Ruzhong be fixed in the emulsion effectively, sanitas can solve short period of time (in the 24 hours) preservation of weather sample in hot period, and emulsifying agent makes the butterfat around the somatocyte of centrifugal back slough through 40 ℃ of constant temperature 5-10 minutes.
Although the somatocyte in the milk through purifying, has other materials in addition around its cytolemma, therefore when lysing cell, add emulsifying agent once more and remove somatocyte butterfat on every side, improve the concentration and the usage quantity of lysate part reagent, it is more thorough that somatocyte is cracked, to obtain more DNA.
Through the washing precipitation of ethanol concn gradient, proteinaceous substances is thoroughly separated behind total DNA that effectively will be wherein and the partially denaturing, also can help to clean simultaneously DNA, removes impurity after digested overnight.
Because the Ruzhong protein content is higher, therefore the present invention takes phenol, chloroform/primary isoamyl alcohol and the chloroformic solution of two volumes that sample mix liquid has been carried out the single extracting, repeat extractive conventional thinking but not carry out equal-volume, saved the time effectively, improved extraction rate was acquired simultaneously.
The effect of the cow blood genomic dna that present method and ordinary method (phynol method) are extracted relatively after, find that effect is the same, can utilize the technology of the present invention source of sample of will suckle as the total DNA of extraction.And utilize GHR (growth hormone receptor) gene to carry out pcr amplification and further verify the DNA that is extracted, the result who found that the dna profiling amplification of milk sample source is identical with blood source dna profiling amplification, can be used for molecular biology research so fundamentally proved milk DNA that sample extracts, and the technology of the present invention also is practicable.
Description of drawings
Fig. 1: be to use the inventive method to extract the milk total DNA of sample and utilize ordinary method to extract between the total dna gel electrophoresis result of blood sample that swimming lane M is 100bp ladder Marker in the comparison diagram, 1,2,3 extracts total DNA for blood sample, 4,5,6 for the milk sample extracts total DNA, and 100bp ladder Marker contains bio tech ltd available from new east station of Guangzhou;
Fig. 2: use pcr amplification GHR gene the 9th exon gel swimming lane M Marker I among the figure as a result, 1,2,3,4,5 with milk sample dna profiling amplification GHR the 9th exon fragment, 6,7,8,9 is with blood sample dna profiling amplification GHR the 9th exon fragment, 10 negative contrasts.MarkerI contains bio tech ltd available from new east station of Guangzhou.
Embodiment
1, total DNA extraction comprises from milk the extraction of total DNA in the extraction of total DNA and the cow blood;
(1) extraction of the total DNA of milk sample, step is as follows:
1) prepare before the sampling: clean mammilla of milk cattle with the tincture of iodine, dry the Supramammillary tincture of iodine with clean warm water cloth, the preceding 2-3 that abandons extruding obtains the milk sample to milk with milking apparatus or hand milking's method afterwards;
2) sampling: add 1ml somatocyte stationary liquid and 20% potassium bichromate 0.5ml in advance as sanitas in centrifuge tube, pack the milk sample of taking into centrifuge tube to 50ml, in 2500 rev/mins, 4 ℃ centrifugal 30 minutes;
3) discard step 2) the upper strata butterfat and the middle layer emulsion of centrifuge tube, keep its bottom settlings, add 1ml ditalimfos acid buffer to the bottom, bottom settlings beaten suspend and be transferred in the centrifuge tube of 1.5ml, at 3500 rev/mins, under the normal temperature centrifugal 10 minutes, discard supernatant liquid, keep bottom settlings;
4) in the centrifuge tube of step 3), add the phosphoric acid buffer 1ml of sterilization, the precipitation that suspends, with the vibrator vibration till precipitation suspends fully, then at 1000 rev/mins, under the normal temperature centrifugal 10 minutes;
5) after centrifugal supernatant is discarded, add emulsifying agent 150 μ l, add the phosphoric acid buffer 1350 μ l of sterilization again, suspend fully with vibrator vibration to precipitation, the butterfat of sloughing in 5-10 minute around the somatocyte is handled in 40 ℃ of waters bath with thermostatic control;
6) after the water-bath, at 3500 rev/mins, under the normal temperature centrifugal 10 minutes, abandon supernatant after centrifugal, add the phosphoric acid buffer 1ml precipitation that suspends, mixed solution is transferred in the centrifuge tube of 1.5ml, made the somatocyte precipitation in centrifugal 10 minutes in 12000 rev/mins, with the somatocyte that obtains put-20 ℃ frozen, perhaps directly described somatocyte is carried out DNA extraction;
7) have adding cracking mixed liquor I 600 μ l and cracking mixed liquor I I 150 μ l in the somatic 1.5ml centrifuge tube to step 6), vibration makes its bottom settlings to suspending fully, places 56 ℃ of water-bath digested overnight, obtains digestion product;
8) digestion product to step 7) adds isopyknic dehydrated alcohol, and 12000 rev/mins of centrifugations discard top ethanol, 95%, 75%, 70% ethanol that adds 1ml then respectively carries out the gradient washing, discard the ethanol on upper strata respectively, ethanol evaporation is 5 minutes at normal temperatures, obtains the DNA crude product;
9) the DNA crude product that step 8) is obtained adds 500 μ l deionization aqua sterilisas makes its dissolution precipitation, and the pH that adds two volumes again is that 8.0 the saturated phenol solution of Tris carries out extracting, puts upside down back and forth 10 minutes, 12000 rev/mins, under the normal temperature centrifugal 10 minutes, obtains supernatant liquor;
10) supernatant liquor of step 9) being transferred in another 1.5ml centrifuge tube, put upside down back and forth 10 minutes, is the phenol of 25: 24: 1 adding two volumes: chloroform by volume: primary isoamyl alcohol, and at 12000 rev/mins, extracting 10 minutes obtains supernatant liquor;
11) supernatant liquor of step 10) being transferred in another 1.5ml centrifuge tube, is 24: 1 adding two volumes chloroforms by volume: primary isoamyl alcohol, put upside down back and forth 10 minutes, and 12000 rev/mins, extracting 10 minutes obtains supernatant liquor;
12) supernatant liquor of step 11) is transferred in another 1.5ml centrifuge tube, added the 3M/L of 1/10 volume, pH is the dehydrated alcohol of the precooling of 5.2 NaAc and 2 times of volumes, rocks centrifuge tube back and forth, places 30 minutes in-20 ℃; In 12000 rev/mins, 25 ℃ were descended centrifugal 10 minutes, carefully outwelled the upper strata dehydrated alcohol, obtained the DNA white precipitate again;
13) the ethanol 1ml of adding 70%, washing step 12) described DNA white precipitate, in centrifugal 10 minutes of following 12000 rev/mins of normal temperature, carefully discard ethanol, the residual ethanol of volatilizing at normal temperatures, 50-100 μ l three (methylol) aminomethane-ethylenediamine tetraacetic acid (EDTA) (Tris-EDTA/TE) that the adds dissolving DNA that spends the night obtains the pure product of DNA;
Wherein,
Step 2) described somatocyte stationary liquid is formulated as follows:
35-40% formaldehyde 9.4ml is dissolved in the 100ml distilled water surely;
The described emulsifying agent of step 5) is formulated as follows:
In the 1L emulsifying agent: 90% triton-x 100 20ml, 95% ethanol 125ml, 0.9g/l NaCl solution 855ml;
The described cracking mixed liquor I of step 7) is formulated as follows:
Mixed liquor I: NaCl 1M/L, Tris-Cl 0.5M/L, EDTA-Na 0.5M/L transfers pH to 7.5-8.0;
The described cracking mixed liquor I of step 7) I is formulated as follows:
20% sodium lauryl sulphate, 40 μ l, the Proteinase K 20 μ l of 20mg/ml, emulsifying agent 90 μ l;
The described Tris-EDTA solution of step 13) is formulated as follows:
Get pH respectively and be 8.0 1M/L Tris-Cl 2ml and 0.5M/L EDTA 0.4ml, its two kinds of solution are mixed, adding distil water is settled to 200ml, transfers pH to 8.0.
Step 3), 4), 5) and 6) described phosphoric acid buffer is formulated as follows:
Take by weighing NaCl 8g respectively, KCL 0.2g, Na
2HPO
41.44g, KH
2PO
40.24g, various substance dissolves in 800ml distilled water, are transferred pH to 7.4, adding distil water is settled to 1L.
(2) extract total DNA in the cow blood, concrete grammar reference " molecular cloning experiment guide " third edition, J. Sa nurse Brooker, D.M, Russell's work, Huang Peitang etc. translate.
2, milk sample and cow blood DNA extraction result compare detection
Detection method: prepare 1.2% sepharose, draw 1.5 μ l DNA samples, add 1 μ l sample-loading buffer, electrophoresis is 15 minutes under the 100V voltage stabilizing, and electrophoresis finishes, and puts gel in ultraviolet transilluminator observation down, as reference, detected result is seen accompanying drawing 1. with 100bp ladder
As can be seen from Figure 1, the milk sample dna fragmentation size of being extracted is the same with the DNA that extracts from cow blood, and the banding pattern neat and consistent shows that the milk sample DNA purity of being extracted is higher, does not obviously trail and diffusing phenomenon, shows that the DNA integrity of extraction is relatively good.
Wherein,
Being formulated as follows of sample-loading buffer described in the step 2:
4.17% bromjophenol blue, 4.17% dimethylbenzene green grass or young crops, 2.5%Ficoll ficoll
This example has been taked the new fresh milk sample of 5 cow heads at random from Hui Erkang Yangtze River, Wuhan, Wuhan City, Hubei Province dairy industry company limited Wu Hu pasture, according to the total DNA in the method extraction milk sample of the present invention, after getting 50 times of 4 μ L milk sample DNA dilutions, on Beckman-DU 800 type ultraviolet spectrophotometers, measure OD
260/280Value and DNA concentration.The concentration calculation formula that is adopted is: A260* extension rate * 50ug/ul.Obtain data as shown in table 1:
Table 1 milk sample DNA purity and concentration determination result
| Milk sample numbering | DNA purity (OD 260/280Value) | DNA concentration (ug/ul) |
| 1 | 1.822 | 53.75 |
| 2 | 1.857 | 9.00 |
| 3 | 1.804 | 28.00 |
| 4 | 1.820 | 26.25 |
| 5 | 1.813 | 39.00 |
As can be seen from Table 1, DNA purity OD
260/280Value is within the 1.8-2.0 scope, and its DNA purity meets the requirements.Concentration is outstanding at interindividual variation, and reason is because in the Different Individual milk sample due to the contained somatocyte difference.
PCR detects DNA extraction and purification effect:
This example utilizes total DNA that total DNA that milk extracts and blood extracts as pcr template growth hormone receptor (GHR) gene the 9th exon that increases, comparison and detection expanding effect.
1, primer sequence
Forward primer: 5 '-TAGGAGTTCCTTTTAGAGGATAGGTGC-3 '
Reverse primer: 5 '-GCCTTGTGGAGAAGTTGACAAA-3 '
The primer reference Blott S, Kim J J, Moisio S, et al.Molecular Dissection of a QuantitativeTrait Locus:A PHenylalanine-to-Tyrosine Substitution in the Transmembrane Domain of the BovineGrowth Hormone Receptor Is Associated With a Major Effect on Milk Yield and Composition.J.Genetics, 2003,163:253～266.
2, PCR reaction system
Cumulative volume is to add respectively in the PCR reaction system of 10 μ l:
ddH
2O 7.2μl
10*PCR buffer 1.0μl
10mmol/L dNTPs 0.3μl
2.5U Taq enzyme 0.2 μ l
50mmol/L Mg
2+ 0.4μl
100ng/ μ l forward primer 0.2 μ l
100ng/ μ l reverse primer 0.2 μ l
Dna profiling 0.5 μ l
Cumulative volume 10.0 μ l
3, PCR response procedures
94 ℃ of pre-sex change 5min, then 94 ℃ of sex change 30s, 59.7 ℃ of annealing 30s, 72 ℃ of connection 30s circulate 35 times, and last 72 ℃ are extended 5min, 16 ℃ of insulation 5min.Milk sample and blood sample DNA with extraction are that template is carried out the PCR reaction, amplification GHR gene the 9th exon, and its amplified production clip size is 307bp.
GHR gene the 9th exon sequence that is increased is as follows:
TAGGAGTTCCTTTTAGAGGATAGGTGCAATTTTAGTTTTGAAAAGAAAGAAAAAAGAAGAGAATTTTTGTGAGGTTGGGCCAGGTCACA
TGTTGACATTTTGAAAATGAGAATGTAACAAGGTCACATCAGACCATTTTTCACAGGGGTGACTAATGGGTTTTTTCCCCTCTTTGTCT
TTCAGCCACACCAGCTTTCCTTGTCAGAGCATCTCAGAGTCTGCAGATACTATATCCAGTCCTAGAGACAAGTAAGAATTTCAGTCCTT
TTTCTGCCTTTGGCGATAATGTTCAGATTTTACTGCAGTTCAAGCTACTGTTCTTACTCTGTTTTATTTAGGATATGGTAACTGGATGA
ATTAATAACTGAAATTGTAGGGGCAAACTAAAAAAATTTTTTTTTTCAGAATTTGTCAACTTCTCCACAAGGC
4, the agarose gel electrophoresis of pcr amplification product detects
Detection method: the sepharose of preparation 1.2%, draw 5 μ l PCR products, add 1 μ l sample-loading buffer (with described in embodiment 1 step 2 identical), electrophoresis is 15 minutes under the 100V voltage stabilizing, electrophoresis finishes, and puts gel in down observation of ultraviolet transilluminator, with Marker I as reference, carry out the PCR product and identify that detected result is seen accompanying drawing 2.
The milk sample DNA that extracts with the present invention is a template, and the segmental size of pcr amplification product is 307bp, by shown in Figure 2, can prove that obviously milk sample DNA can successfully amplify GHR gene the 9th exon, expanding effect be that the template amplification result is the same with blood sample DNA.
The explanation of table 2 english abbreviation involved in the present invention
Claims (3)
1, a kind of method of extracting total DNA from milk is characterized in that following steps:
1) prepare before the sampling: clean mammilla of milk cattle with the tincture of iodine, dry the Supramammillary tincture of iodine with clean warm water cloth, the preceding 2-3 that abandons extruding obtains the milk sample to milk with milking apparatus or hand milking's method afterwards;
2) sampling: add 1ml somatocyte stationary liquid and 20% potassium bichromate 0.5ml in advance as sanitas in centrifuge tube, pack the milk sample of taking into centrifuge tube to 50ml, in 2500 rev/mins, 4 ℃ centrifugal 30 minutes;
3) discard step 2) the upper strata butterfat and the middle layer emulsion of centrifuge tube, keep its bottom settlings, add 1ml ditalimfos acid buffer to the bottom, bottom settlings beaten suspend and be transferred in the centrifuge tube of 1.5ml, at 3500 rev/mins, under the normal temperature centrifugal 10 minutes, discard supernatant liquid, keep bottom settlings;
4) in the centrifuge tube of step 3), add the phosphoric acid buffer 1ml of sterilization, the precipitation that suspends, with the vibrator vibration till precipitation suspends fully, then at 1000 rev/mins, under the normal temperature centrifugal 10 minutes;
5) after centrifugal supernatant is discarded, add emulsifying agent 150 μ l, add the phosphoric acid buffer 1350 μ l of sterilization again, suspend fully with vibrator vibration to precipitation, the butterfat of sloughing in 5-10 minute around the somatocyte is handled in 40 ℃ of waters bath with thermostatic control;
6) after the water-bath, at 3500 rev/mins, under the normal temperature centrifugal 10 minutes, abandon supernatant after centrifugal, add the phosphoric acid buffer 1ml precipitation that suspends, mixed solution is transferred in the centrifuge tube of 1.5ml, made the somatocyte precipitation in centrifugal 10 minutes in 12000 rev/mins, with the somatocyte that obtains put-20 ℃ frozen, perhaps directly described somatocyte is carried out DNA extraction;
7) have adding cracking mixed liquor I 600 μ l and cracking mixed liquor I I 150 μ l in the somatic 1.5ml centrifuge tube to step 6), vibration makes its bottom settlings to suspending fully, places 56 ℃ of water-bath digested overnight, obtains digestion product;
8) digestion product to step 7) adds isopyknic dehydrated alcohol, and 12000 rev/mins of centrifugations discard top ethanol, 95%, 75%, 70% ethanol that adds 1ml then respectively carries out the gradient washing, discard the ethanol on upper strata respectively, ethanol evaporation is 5 minutes at normal temperatures, obtains the DNA crude product;
9) the DNA crude product that step 8) is obtained adds 500 μ l deionization aqua sterilisas makes its dissolution precipitation, and the pH that adds two volumes again is that 8.0 the saturated phenol solution of Tris carries out extracting, puts upside down back and forth 10 minutes, 12000 rev/mins, under the normal temperature centrifugal 10 minutes, obtains supernatant liquor;
10) supernatant liquor of step 9) being transferred in another 1.5ml centrifuge tube, put upside down back and forth 10 minutes, is the phenol of 25: 24: 1 adding two volumes: chloroform by volume: primary isoamyl alcohol, and at 12000 rev/mins, extracting 10 minutes obtains supernatant liquor;
11) supernatant liquor of step 10) being transferred in another 1.5ml centrifuge tube, is 24: 1 adding two volumes chloroforms by volume: primary isoamyl alcohol, put upside down back and forth 10 minutes, and 12000 rev/mins, extracting 10 minutes obtains supernatant liquor;
12) supernatant liquor of step 11) is transferred in another 1.5ml centrifuge tube, added the 3M/L of 1/10 volume, pH is the dehydrated alcohol of the precooling of 5.2 NaAc and 2 times of volumes, rocks centrifuge tube back and forth, places 30 minutes in-20 ℃; In 12000 rev/mins, 25 ℃ were descended centrifugal 10 minutes, carefully outwelled the upper strata dehydrated alcohol, obtained the DNA white precipitate again;
13) the ethanol 1ml of adding 70%, washing step 12) described DNA white precipitate, in centrifugal 10 minutes of following 12000 rev/mins of normal temperature, carefully discard ethanol, the residual ethanol of volatilizing at normal temperatures, 50-100 μ l three (methylol) aminomethane-ethylenediamine tetraacetic acid (EDTA) that the adds dissolving DNA that spends the night obtains the pure product of DNA;
Wherein,
Step 2) described somatocyte stationary liquid is formulated as follows:
35-40% formaldehyde 9.4ml is dissolved in the 100ml distilled water surely;
The described emulsifying agent of step 5) is formulated as follows:
In the 1L emulsifying agent: 90% Triton X-100 (triton-x 100) 20ml, 95% ethanol 125ml, 0.9g/l NaCl solution 855ml;
The described cracking mixed liquor I of step 7) is formulated as follows:
Mixed liquor I: NaCl 1M/L, Tris-Cl 0.5M/L, EDTA-Na 0.5M/L transfers pH to 7.5-8.0;
The described cracking mixed liquor I of step 7) I is formulated as follows:
20% sodium lauryl sulphate, 40 μ l, the Proteinase K 20 μ l of 20mg/ml, emulsifying agent 90 μ l;
The described Tris-EDTA solution of step 13) is formulated as follows:
Get pH respectively and be 8.0 1M/L Tris-Cl 2ml and 0.5M/L EDTA 0.4ml, its two kinds of solution are mixed, adding distil water is settled to 200ml, transfers pH to 8.0.
Step 3), 4), 5) and 6) described phosphoric acid buffer is formulated as follows:
Take by weighing NaCl 8g respectively, KCL 0.2g, Na
2HPO
41.44g, KH
2PO
40.24g, various substance dissolves in 800ml distilled water, are transferred pH to 7.4, adding distil water is settled to 1L.
2, method according to claim 1 is characterized in that, used milk sample be behind the adding preservative agent 24 hours with interior milk sample.
3, the application of the described method of claim 1 in the operation of milk cow molecular biology.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559659A (en) * | 2011-12-28 | 2012-07-11 | 浙江工业大学 | Method for purifying DNA (deoxyribonucleic acid ) by utilizing ethanol fractional precipitation |
| CN102719426A (en) * | 2012-06-08 | 2012-10-10 | 陕西师范大学 | Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification |
| CN103882012A (en) * | 2014-04-21 | 2014-06-25 | 山东省农业科学院生物技术研究中心 | Method for rapidly and efficiently extracting DNA (Desoxvribose Nucleic Acid) from milk products such as liquid milk and milk powder |
| CN105018606A (en) * | 2015-07-07 | 2015-11-04 | 陕西师范大学 | DNA quality based method for evaluation of milk freshness |
| CN105018607A (en) * | 2015-07-07 | 2015-11-04 | 陕西师范大学 | DNA quality-based method for identification of fat and protein content in milk |
| CN108396025A (en) * | 2018-01-23 | 2018-08-14 | 安徽微分基因科技有限公司 | A method of extracting microorganism MetaDNA from lacto's sample |
| CN110437988A (en) * | 2019-07-30 | 2019-11-12 | 河南省奶牛生产性能测定中心 | A kind of extract separating pipe and its application method |
| CN110527681A (en) * | 2018-05-25 | 2019-12-03 | 中国农业科学院北京畜牧兽医研究所 | The extracting method of total microbial DNA in a kind of milk |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3781577B2 (en) * | 1999-03-17 | 2006-05-31 | 雪印乳業株式会社 | RNA extraction and long-term storage method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559659A (en) * | 2011-12-28 | 2012-07-11 | 浙江工业大学 | Method for purifying DNA (deoxyribonucleic acid ) by utilizing ethanol fractional precipitation |
| CN102559659B (en) * | 2011-12-28 | 2013-07-31 | 浙江工业大学 | Method for purifying DNA (deoxyribonucleic acid ) by utilizing ethanol fractional precipitation |
| CN102719426A (en) * | 2012-06-08 | 2012-10-10 | 陕西师范大学 | Method for extracting genomic DNA (deoxyribonucleic acid) in milk appropriate for large-scale genotype identification |
| CN103882012A (en) * | 2014-04-21 | 2014-06-25 | 山东省农业科学院生物技术研究中心 | Method for rapidly and efficiently extracting DNA (Desoxvribose Nucleic Acid) from milk products such as liquid milk and milk powder |
| CN103882012B (en) * | 2014-04-21 | 2016-10-05 | 山东省农业科学院生物技术研究中心 | A kind of quickly method of high efficiency extraction DNA from the milk product such as cattle and sheep liquid milk and milk powder |
| CN105018606A (en) * | 2015-07-07 | 2015-11-04 | 陕西师范大学 | DNA quality based method for evaluation of milk freshness |
| CN105018607A (en) * | 2015-07-07 | 2015-11-04 | 陕西师范大学 | DNA quality-based method for identification of fat and protein content in milk |
| CN105018607B (en) * | 2015-07-07 | 2018-10-26 | 陕西师范大学 | A method of based on Oil content and Protein content in DNA Quality Identification milk |
| CN105018606B (en) * | 2015-07-07 | 2018-10-30 | 陕西师范大学 | A method of based on DNA Quality Identification milk freshness |
| CN108396025A (en) * | 2018-01-23 | 2018-08-14 | 安徽微分基因科技有限公司 | A method of extracting microorganism MetaDNA from lacto's sample |
| CN110527681A (en) * | 2018-05-25 | 2019-12-03 | 中国农业科学院北京畜牧兽医研究所 | The extracting method of total microbial DNA in a kind of milk |
| CN110437988A (en) * | 2019-07-30 | 2019-11-12 | 河南省奶牛生产性能测定中心 | A kind of extract separating pipe and its application method |
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