CN104450676A - Method for extracting DNA from animal tissue efficiently - Google Patents
Method for extracting DNA from animal tissue efficiently Download PDFInfo
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- CN104450676A CN104450676A CN201410414369.7A CN201410414369A CN104450676A CN 104450676 A CN104450676 A CN 104450676A CN 201410414369 A CN201410414369 A CN 201410414369A CN 104450676 A CN104450676 A CN 104450676A
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Abstract
The invention discloses a method for extracting DNA from an animal tissue efficiently. The method comprises the following steps of (1) selecting an animal tissue sample, and adding a buffer solution A and proteinase K solution into the animal tissue sample to obtain a mixture; (2) putting the mixture into incubation equipment, adjusting the temperature of the incubation equipment to 55-58 DEG C, and incubating the mixture for 2-5 hours to obtain a sample suspension; (3) oscillating the sample suspension, adding sodium chloride solution, mixing the sample suspension with the sodium chloride solution uniformly, and centrifuging to obtain a supernatant; (4) adding isopropanol into the supernatant, mixing the isopropanol and the supernatant uniformly, centrifuging, and removing the supernatant to obtain a precipitate; and (5) removing the residual isopropanol in the precipitate by washing with aqueous solution of ethanol, centrifuging, and standing to remove ethanol to obtain a precipitate which is the DNA of the animal tissue. The yield of DNA extracted from the animal tissue sample by adopting the method is high, and the method is simple in step, is harmless to the human body, can not cause pollution to environment and can be used for extracting a large amount of DNAs from the animal tissue samples rapidly.
Description
Technical field
The invention belongs to DNA extraction technical field, be specifically related to a kind of method of high efficiency extraction animal tissues DNA.
Background technology
Containing a large amount of DNA in animal tissues, DNA is chromosomal main chemical compositions, is also the material of constitutive gene simultaneously.The function of DNA molecular is that storage determines nearly all protein of species proterties and whole genetic information of RNA molecule, coding and design biological organism aerial open gene and expressing protein in an orderly manner when certain complete directed all programs of growing, tentatively determine biological exclusive proterties and individual character and and environmental interaction time all stress reaction.Therefore by research to DNA, as the situations such as DNA methylation, base deletion, sudden change, the physiological status can indirectly understanding or evaluate animal body and response situation to external world.And set up efficiently, accurately DNA method for extracting also become a key problem in technology place of animal molecular mechanism research.
The major objective of the extraction of animal tissues DNA is that the DNA obtaining most high-content and highest purity reclaims, and application DNA is studied by various method as research object further.But animal tissues forms complicated, comprises lipid, protein, carbohydrate, VITAMIN, mineral element etc., has impact to the extraction of DNA, especially lipid, protein and DNA weave in, be difficult to remove totally.In addition, after lysis, each step of extracting DNA can have the loss of DNA, and therefore step is more, and the probability of loss is larger.In addition, in the method mostly continued to use at present, all relate to the application of phenol/chloroform reagent, easily cause environmental pollution, also have harm to the HUMAN HEALTH of experimental implementation person.And operation steps is complicated, DNA yield is low, is difficult to Rapid extraction great amount of samples.
Therefore, need research to be applicable to convenient, fast, the practical method of animal tissues's DNA extraction, low to solve the DNA sample yield using ordinary method to obtain, the problems such as complex operation step.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method for high efficiency extraction animal tissues DNA, and the method extracting animal tissues sample DNA yield is high, and step is simple, and to human body without harm, environmentally safe, can Rapid extraction great amount of samples.
Technical problem to be solved by this invention is realized by following technical problem: a kind of method of high efficiency extraction animal tissues DNA, comprises the following steps:
(1) choose animal tissues's sample, add buffer A and Proteinase K Solution, obtain mixture;
(2) mixture is placed in the equipment of hatching, regulates temperature to be 55 ~ 58 DEG C, hatch 2 ~ 5h, obtain sample suspension liquid;
(3) after being shaken by sample suspension liquid, sodium chloride solution is added, mixing, centrifugal, get supernatant liquor;
(4) in the supernatant liquor of step (3) gained, Virahol is added, mixing, centrifugal, remove supernatant liquor, be precipitated thing;
(5) adopt aqueous ethanolic solution to wash away Virahol residual in throw out, centrifugal rear standing removing ethanol, gained throw out is animal tissues DNA.
Buffer A described in step (1) is by 30 ~ 80mM Tris, 50 ~ 200mM EDTA, 50 ~ 200mM NaCl, 1 ~ 3%(quality volumn concentration) sodium laurylsulfonate SDS forms, and its pH is 7.5 ~ 8.0.
In this buffer A, each component has following effect:
Because DNA is the most stable in weak alkaline environment, therefore the pH value of buffer A is of value to the stable of DNA, and the effect of Tris mainly forms certain weakly alkaline damping fluid with NaCl, maintains pH value of solution balance.
Chelating Mg in EDTA
2+, Ca
2+deng metal ion, deoxyribonuclease can be suppressed the Degradation (needing certain metal ion to make prothetic group during DNase effect) of DNA.
NaCl mainly forms certain weakly alkaline damping fluid with Tris, maintain pH value of solution balance, provide certain salt ionic concentration, maintain the ambient stable residing for DNA.
SDS is ionogenic surfactant, and its major function has: the lipid on (1) dissolved cell film and albumen, thus dissolving film albumen and destroy cytolemma; (2) nucleoprotein in depolymerization cell; (3) SDS can become R-O-SO3-with protein bound ... the mixture of R+-protein, makes protein denaturation and precipitates.
The effect of Proteinase K is digestible protein, due to DNA and protein weave in, needs after proteopepsis, and DNA is free out, just can carry out extracting.
In step (1) animal tissues's sample, buffer A and Proteinase K Solution be preferably μ L:2 ~ 4,5 ~ 10mg:50 ~ 100 μ L with magnitude relation, wherein the concentration of Proteinase K Solution is 10 ~ 20mg/mL.
In step (3), the concentration of sodium chloride solution is preferably 3 ~ 6 molL
-1, the volume ratio of itself and sample suspension liquid is preferably 1:2 ~ 4.
Be in the solution of about 8 at pH, DNA molecular is electronegative, add certain density NaCl, to make in Na+ and negative charge on DNA molecular, reduce the same sex electric charge mutual expelling force between DNA molecular, be easy to polymerization mutually and form DNA sodium salt precipitation, when the concentration of salt solution added is too low, only have part DNA form DNA sodium salt and be polymerized, so just cause DNA to precipitate not exclusively.Therefore the sodium chloride solution of high density is conducive to removing protein, precipitation DNA.
Condition optimization time centrifugal in step (3) is the centrifugal 10 ~ 15min of 10000 ~ 14000rpm under 4 DEG C of conditions.
In step (4), the volume ratio of Virahol and supernatant liquor is preferably 1:1.2 ~ 1.6.The effect of Virahol: protein is thoroughly separated with DNA.
Condition optimization time centrifugal in step (4) is the centrifugal 15 ~ 20min of 10000 ~ 14000rpm under 4 DEG C of conditions.
The volumn concentration of the aqueous ethanolic solution described in step (5) is preferably 70 ~ 75%.
Condition time centrifugal in step (5) is the centrifugal 5 ~ 10min of 10000 ~ 14000rpm under 4 DEG C of conditions.
The animal tissues DNA that the present invention obtains can add H
2o, mixing, preserves under being placed in-20 DEG C of conditions or is directly used in DNA analysis.
Tool of the present invention has the following advantages:
(1) DNA extraction method in the present invention is simple to operate, and do not need comparatively complicated equipment, conventional biochemical laboratory just can complete; And the reagent that the reagent related to is routine analysis, molecular biology uses is low relative to the test kit cost on market;
(2) the DNA extraction method suitability in the present invention is strong, and the OD260/OD280 of the DNA of extraction is near the mark value, and can directly apply to molecule manipulation, effect is better;
(3) the DNA extraction method leaching process in the present invention does not use phenol, chloroform, and reduce the healthy injury to experimenter, the DNA that obtains is comparatively complete in institute, output is higher.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in further detail.
The reagent such as Tris (tricarboxymethyl aminomethane), Tris, HCl, EDTA (disodium ethylene diamine tetraacetate), NaCl, ethanol, SDS (sodium lauryl sulphate), Virahol is domestic analytical pure medicine, and Proteinase K derives from sigma company.
embodiment 1
extract the muscle tissue DNA of pig
(1) muscle tissue sample of clip 100mg pig, add 750 μ L buffer A, 37.5 μ L mass concentrations are the Proteinase K Solution of 10mg/mL, wherein buffer A is by 30mM Tris, 50mM EDTA, 50mM NaCl, 1%(quality volumn concentration) sodium laurylsulfonate SDS forms, and its pH is 7.5 ~ 8.0;
(2) said mixture is placed in 55 ~ 58 DEG C of couveuses (water-bath or miniature electric hatch equipment) 3 hours, obtains sample suspension liquid;
(3) shake about 1 minute, in sample suspension liquid, add 250 μ L concentration is 6 molL
-1sodium chloride solution, mixing, centrifugal 10 minutes of 14000rpm under 4 DEG C of conditions;
(4) supernatant liquor 750 μ L is shifted in new centrifuge tube;
(5) in supernatant liquor, add 500 μ L Virahols, firmly mix (whirlpool shakes 1 minute), under 4 DEG C of conditions, centrifugal 20 minutes of 14000rpm, removes supernatant liquor, obtains throw out;
(6) add 1mL volumn concentration be 75% aqueous ethanolic solution to wash in pipe remaining Virahol off, centrifugal 10 minutes of 14000rpm under 4 DEG C of conditions;
(7) ambient temperatare is put, and allows ethanol volatilize clean, is the muscle tissue DNA of the pig be separated to bottom pipe;
(8) 100 μ LH are added
2o, mixing, by spectrophotometric determination DNA concentration and OD260/OD280 value, result is as follows:
The DNA that table 1 the present embodiment method obtains and the contrast with current DNA extraction agent box on the market
| Index | Concentration (ng/ μ L) | OD260/280 | OD260/230 |
| DNA extraction agent box on the market | 33.6 | 1.86 | 2.05 |
| Method of the present invention | 427.4 | 1.96 | 2.06 |
As can be seen from Table 1, OD260/OD280 standard value is 1.96, the standard value 2.06 of OD260/OD230, method of the present invention is compared with the test kit of commercial type, and OD260/OD280 standard value and OD260/OD230 standard value are close to the test value of DNA on the market, but the concentration of DNA extracting improves greatly, and DNA purity is higher, similar to by the purity of commercially available reagent box extracting DNA, with standard value relatively, can be used for DNA analysis.
embodiment 2 extracts the liver organization DNA of pig
(1) liver tissue sample of clip 50mg pig, add 750 μ L buffer A, 21 μ L mass concentrations are the Proteinase K Solution of 20mg/mL, obtain mixture, wherein buffer A is made up of 50mM Tris, 100mM EDTA, 100mM NaCl, 2% sodium laurylsulfonate SDS, and its pH is 7.5 ~ 8.0;
(2) said mixture is placed in 55 ~ 58 DEG C of couveuses (water-bath or miniature electric hatch equipment) 3 hours, obtains sample suspension liquid;
(3) shake about 1 minute, in sample suspension liquid, add 250 μ L concentration is 3molL
-1sodium chloride solution, mixing, centrifugal 15 minutes of 14000rpm under 4 DEG C of conditions;
(4) supernatant liquor 750 μ L is shifted in new centrifuge tube;
(5) in supernatant liquor, add 500 μ L Virahols, firmly mix (whirlpool shakes 1 minute), under 4 DEG C of conditions, centrifugal 15 minutes of 14000rpm, removes supernatant liquor, obtains throw out;
(6) add 1mL volumn concentration be 75% aqueous ethanolic solution to wash in pipe remaining Virahol off, centrifugal 10 minutes of 14000rpm under 4 DEG C of conditions;
(7) ambient temperatare is put, and allows ethanol volatilize clean, is the liver organization DNA of the pig be separated to bottom pipe;
(8) 100 μ LH are added
2o, mixing, by spectrophotometric determination DNA concentration and OD260/OD280 value, result is as following table 2:
The DNA that table 2 the inventive method is extracted and the Comparative result with current commodity DNA extraction agent box
| Index | Concentration (ng/ μ L) | OD260/280 | OD260/230 |
| DNA extraction agent box on the market | 31.3 | 1.93 | 2.12 |
| Method of the present invention | 1927.1 | 1.89 | 2.11 |
As can be seen from Table 2, OD260/OD280 standard value is 1.89, the standard value 2.11 of OD260/OD230, method of the present invention is compared with the test kit of commercial type, and OD260/OD280 standard value and OD260/OD230 standard value are close to the test value of DNA on the market, but the concentration of DNA extracting improves greatly, and DNA purity is higher, similar to by the purity of commercially available reagent box extracting DNA, with standard value relatively, can be used for DNA analysis.
embodiment 3 extracts the fatty tissue DNA of pig
(1) adipose tissue sample of clip 100mg pig, add 750 μ L buffer A, 30 μ L mass concentrations are the Proteinase K Solution of 15mg/mL, and wherein buffer A is made up of 80mM Tris, 200mM EDTA, 200mM NaCl, 3% sodium laurylsulfonate SDS, and its pH is 7.5 ~ 8.0;
(2) said mixture is placed in 55 ~ 58 DEG C of couveuses (water-bath or miniature electric hatch equipment) 3 hours, obtains sample suspension liquid;
(3) shake about 1 minute, in sample suspension liquid, add 250 μ L concentration is 4molL
-1sodium chloride solution, mixing, centrifugal 15 minutes of 14000rpm under 4 DEG C of conditions;
(4) supernatant liquor 750 μ L is shifted in new centrifuge tube;
(5) in supernatant liquor, add 500 μ L Virahols, firmly mix (whirlpool shakes 1 minute), under 4 DEG C of conditions, centrifugal 20 minutes of 13000rpm, removes supernatant liquor, obtains throw out;
(6) add 1mL volumn concentration be 75% aqueous ethanolic solution to wash in pipe remaining Virahol off, centrifugal 8 minutes of 14000rpm under 4 DEG C of conditions;
(7) ambient temperatare is put, and allows ethanol volatilize clean, is the fatty tissue DNA of the pig be separated to bottom pipe;
(8) 100 μ LH are added
2o, mixing, by spectrophotometric determination DNA concentration and OD260/OD280 value, result is as shown in table 3 below:
The DNA that table 3 the inventive method is extracted and the Comparative result with current commodity DNA extraction agent box
| Index | Concentration (ng/ μ L) | OD260/280 | OD260/230 |
| DNA extraction agent box on the market | 27.3 | 1.86 | 2.04 |
| Method of the present invention | 773.5 | 1.91 | 2.11 |
As can be seen from Table 3, OD260/OD280 standard value is 1.91, the standard value 2.11 of OD260/OD230, method of the present invention is compared with the test kit of commercial type, and OD260/OD280 standard value and OD260/OD230 standard value are close to the test value of DNA on the market, but the concentration of DNA extracting improves greatly, and DNA purity is higher, similar to by the purity of commercially available reagent box extracting DNA, with standard value relatively, can be used for DNA analysis.
embodiment 4 extracts the ovary tissue DNA of duck
(1) the ovary tissue sample of clip 50mg duck, add 1000 μ L buffer A, 20 μ L mass concentrations are the Proteinase K Solution of 20mg/mL, and wherein buffer A is made up of 60mM Tris, 150mM EDTA, 120mM NaCl, 1.5% sodium laurylsulfonate SDS, and its pH is 7.5 ~ 8.0;
(2) said mixture is placed in 55 ~ 58 DEG C of couveuses (water-bath or miniature electric hatch equipment) 5 hours, obtains sample suspension liquid;
(3) shake about 1 minute, in sample suspension liquid, add 250 μ L concentration is 5molL
-1sodium chloride solution, mixing, centrifugal 10 minutes of 14000rpm under 4 DEG C of conditions;
(4) supernatant liquor 800 μ L is shifted in new centrifuge tube;
(5) in supernatant liquor, add 500 μ L Virahols, firmly mix (whirlpool shakes 1 minute), under 4 DEG C of conditions, centrifugal 20 minutes of 14000rpm, removes supernatant liquor, obtains throw out;
(6) add 1mL volumn concentration be 70% aqueous ethanolic solution to wash in pipe remaining Virahol off, centrifugal 10 minutes of 14000rpm under 4 DEG C of conditions;
(7) ambient temperatare is put, and allows ethanol volatilize clean, is the fatty tissue DNA of the pig be separated to bottom pipe;
(8) 100 μ LH are added
2o, mixing, by spectrophotometric determination DNA concentration and OD260/OD280 value, result is as shown in table 3 below:
The DNA that table 4 the inventive method is extracted and the Comparative result with current commodity DNA extraction agent box
| Index | Concentration (ng/ μ L) | OD260/280 | OD260/230 |
| DNA extraction agent box on the market | 26.0 | 1.87 | 2.01 |
| Method of the present invention | 839.5 | 1.90 | 2.04 |
As can be seen from Table 4, OD260/OD280 standard value is 1.90, the standard value 2.04 of OD260/OD230, method of the present invention is compared with the test kit of commercial type, and OD260/OD280 standard value and OD260/OD230 standard value are close to the test value of DNA on the market, but the concentration of DNA extracting improves greatly, and DNA purity is higher, similar to by the purity of commercially available reagent box extracting DNA, with standard value relatively, can be used for DNA analysis.
embodiment 5 extracts the renal tissue DNA of chicken
(1) the renal tissue sample of clip 80mg chicken, add 60 μ L buffer A, 35 μ L mass concentrations are the Proteinase K Solution of 15mg/mL, and wherein buffer A is made up of 40mM Tris, 90mM EDTA, 90mM NaCl, 2.5% sodium laurylsulfonate SDS, and its pH is 7.5 ~ 8.0;
(2) said mixture is placed in 55 ~ 58 DEG C of couveuses (water-bath or miniature electric hatch equipment) 2 hours, obtains sample suspension liquid;
(3) shake about 1 minute, in sample suspension liquid, add 250 μ L μ L concentration is 4molL
-1sodium chloride solution, mixing, centrifugal 12 minutes of 14000rpm under 4 DEG C of conditions;
(4) supernatant liquor 600 μ L is shifted in new centrifuge tube;
(5) in supernatant liquor, add 500 μ L Virahols, firmly mix (whirlpool shakes 1 minute), under 4 DEG C of conditions, centrifugal 15 minutes of 14000rpm, removes supernatant liquor, obtains throw out;
(6) add 1mL volumn concentration be 72% aqueous ethanolic solution to wash in pipe remaining Virahol off, centrifugal 8 minutes of 14000rpm under 4 DEG C of conditions;
(7) ambient temperatare is put, and allows ethanol volatilize clean, is the fatty tissue DNA of the pig be separated to bottom pipe;
(8) 100 μ LH are added
2o, mixing, by spectrophotometric determination DNA concentration and OD260/OD280 value, result is as shown in table 3 below:
The DNA that table 5 the inventive method is extracted and the Comparative result with current commodity DNA extraction agent box
| Index | Concentration (ng/ μ L) | OD260/280 | OD260/230 |
| DNA extraction agent box on the market | 32.5 | 1.90 | 2.03 |
| Method of the present invention | 635.4 | 1.96 | 2.10 |
As can be seen from Table 5, OD260/OD280 standard value is 1.96, the standard value 2.10 of OD260/OD230, method of the present invention is compared with the test kit of commercial type, and OD260/OD280 standard value and OD260/OD230 standard value are close to the test value of DNA on the market, but the concentration of DNA extracting improves greatly, and DNA purity is higher, similar to by the purity of commercially available reagent box extracting DNA, with standard value relatively, can be used for DNA analysis.
Above-described embodiment is the present invention's preferably embodiment, but embodiments of the present invention are not restricted to the described embodiments, if the animal tissues's sample in the present invention is that effect is identical substantially, as the muscle tissue of pig and mouse, rat, chicken, duck, the muscle tissue extraction rate was acquired of ox, sheep, rabbit etc. is the same; The fatty tissue of pig and mouse, rat, chicken, duck, the fatty tissue extraction rate was acquired of ox, sheep, rabbit etc. is the same; The liver organization of pig is the same with the extraction rate was acquired of other animal livers tissue; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included in protection scope of the present invention.
Claims (9)
1. a method of high efficiency extraction animal tissues DNA, is characterized in that comprising the following steps:
(1) choose animal tissues's sample, add buffer A and Proteinase K Solution, obtain mixture;
(2) mixture is placed in the equipment of hatching, regulates temperature to be 55 ~ 58 DEG C, hatch 2 ~ 5h, obtain sample suspension liquid;
(3) after being shaken by sample suspension liquid, sodium chloride solution is added, mixing, centrifugal, get supernatant liquor;
(4) in the supernatant liquor of step (3) gained, Virahol is added, mixing, centrifugal, remove supernatant liquor, obtain precipitation;
(5) adopt aqueous ethanolic solution to wash away Virahol residual in precipitation, centrifugal rear standing removing ethanol, gained throw out is animal tissues DNA.
2. the method for high efficiency extraction animal tissues DNA according to claim 1, it is characterized in that: the buffer A described in step (1) is that 1 ~ 3% sodium laurylsulfonate SDS forms by 30 ~ 80mM Tris, 50 ~ 200mM EDTA, 50 ~ 200mM NaCl and quality volumn concentration, and its pH is 7.5 ~ 8.0.
3. the method for high efficiency extraction animal tissues DNA according to claim 2, it is characterized in that: in step (1), animal tissues's sample, buffer A and Proteinase K Solution is μ L:2 ~ 4,5 ~ 10mg:50 ~ 100 μ L with magnitude relation, and wherein the concentration of Proteinase K Solution is 10 ~ 20mg/mL.
4. the method for high efficiency extraction animal tissues DNA according to claim 1, is characterized in that: in step (3), the concentration of sodium chloride solution is 3 ~ 6 molL
-1.
5. the method for high efficiency extraction animal tissues DNA according to claim 1, is characterized in that: condition time centrifugal in step (3) is the centrifugal 10 ~ 15min of 10000 ~ 14000rpm under 4 DEG C of conditions.
6. the method for high efficiency extraction animal tissues DNA according to claim 1, is characterized in that: in step (4), the volume ratio of Virahol and supernatant liquor is 1:1.2 ~ 1.6.
7. the method for high efficiency extraction animal tissues DNA according to claim 1, is characterized in that: condition time centrifugal in step (4) is the centrifugal 15 ~ 20min of 10000 ~ 14000rpm under 4 DEG C of conditions.
8. the method for high efficiency extraction animal tissues DNA according to claim 1, is characterized in that: the volumn concentration of the aqueous ethanolic solution described in step (5) is 70 ~ 75%.
9. the method for high efficiency extraction animal tissues DNA according to claim 1, is characterized in that: condition time centrifugal in step (5) is the centrifugal 5 ~ 10min of 10000 ~ 14000rpm under 4 DEG C of conditions.
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| CN112143768A (en) * | 2020-09-29 | 2020-12-29 | 北京赛升药业股份有限公司 | Method for jointly preparing DNA and thymosin by using calf thymus |
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Application publication date: 20150325 |