CN104830836A - Method for extracting tupaia belangeri tissue DNA - Google Patents
Method for extracting tupaia belangeri tissue DNA Download PDFInfo
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Abstract
The invention discloses a method for extracting tupaia belangeri tissue DNA. The method comprises the following steps: (a) centrifuging the tupaia belangeri tissue in a homogenate; (b) centrifuging the liquid supernatant obtained in the step (a); (c) centrifuging the sediment obtained in the step (b) in the homogenate; (d) uniformly mixing the sediment obtained in the step (c) with a first solution, mixing with a second solution and dissolving; (e) mixing the solution obtained in the step (d) with a third solution and performing ice dissolving; (f) centrifuging the solution obtained in the step (e); (g) extracting the liquid supernatant obtained in the step (f) in the mixed solution, and repeating once; (h) mixing the liquid supernatant obtained in the step (g) with the absolute ethyl alcohol, and performing overnight aging; and (i) centrifuging the solution obtained in the step (h), and washing and drying by ethanol. By restricting the reagent types, the reagent dosages and the extraction conditions in the extraction process, the purity of the extracted DNA is high, the electrophoretic band does not have tailing phenomena and the cost is low.
Description
Technical field
The invention belongs to DNA extraction technical field, particularly relate to a kind of extracting method of middle remote tree shrew tissue DNA.
Background technology
Middle remote tree shrew is small-sized type of the climbing up by holding on to Mammals of a class, likeness in form squirrel, conventional laboratory rodent such as comparatively mouse, rat etc. is fastened closer to the mankind in kind, and many diseases similar to people can be suffered from, at present except chimpanzee, gibbon, uniquely can infect the animal of human hepatitis B virus, receive showing great attention to of biology and medical worker.
When the remote tree shrew of centering is studied, extract its tissue DNA and to be absolutely necessary one of step.Prior art discloses the extracting method of other animal DNAs many, but these methods are not all suitable for the extraction that middle remote tree shrew organizes NDA, extract the DNA purity obtained lower, the electrophoretic band conditions of streaking of DNA is serious.
Summary of the invention
The object of the present invention is to provide a kind of extracting method of middle remote tree shrew tissue DNA, it is higher that extracting method provided by the invention extracts the DNA purity obtaining middle remote tree shrew, and electrophoretic band is without conditions of streaking.
The invention provides a kind of extracting method of middle remote tree shrew tissue DNA, comprise the following steps:
A) be organized in by middle remote tree shrew at 3 ~ 5 DEG C, centrifugal 8min ~ 12min under 1200r/min ~ 1800r/min condition in homogenate, the mass volume ratio of described middle remote tree shrew tissue and homogenate is 0.15 ~ 0.25g:0.5 ~ 1.5mL;
B) supernatant liquor step a) obtained is at 3 ~ 5 DEG C, centrifugal 18min ~ 22min under 1200r/min ~ 1800r/min condition;
C) by step c) being deposited at 3 ~ 5 DEG C, centrifugal 8min ~ 12min under 1200r/min ~ 1800r/min condition in homogenate of obtaining, described homogenate is 1.5 ~ 2.5mL:0.15 ~ 0.25g with the volume mass ratio of middle remote tree shrew tissue;
D) by step c) precipitation that obtains mixes with the first solution, the more molten 8min ~ 12min of ice after mixing with the second solution; Described first solution comprises Tris-HCl, Na
2eDTA and NaCl; Described second solution comprises NaOH and sodium lauryl sulphate; The volume mass ratio of described first solution, the second solution and middle remote tree shrew tissue is 40 ~ 60 μ L:90 ~ 110 μ L:0.15 ~ 0.25g;
E) by steps d) solution that obtains mixes with the 3rd solution, the molten 50min ~ 70min of ice; Described 3rd solution comprises potassium ion and acetic acid ion; The volume ratio of described 3rd solution and the second solution is 70 ~ 80:90 ~ 110;
F) by step e) solution centrifugal 8min ~ 12min under 11000r/min ~ 13000r/min condition of obtaining;
G) by step f) supernatant liquor that obtains in mixing solutions under 11000r/min ~ 13000r/min condition extracting 2 ~ 3min, repeat once; Described mixing solutions comprises phenol, chloroform and primary isoamyl alcohol; The volume ratio of described supernatant liquor and described mixing solutions is 0.8 ~ 1.2:0.8 ~ 1.2;
H) by step g) supernatant liquor that obtains mixes with the dehydrated alcohol of 3 DEG C ~ 5 DEG C, spends the night-35 DEG C ~-45 DEG C placements;
I) by step h) solution that obtains at 3 DEG C ~ 5 DEG C, centrifugal 15min ~ 30min under 11000r/min ~ 13000r/min, with 3 DEG C ~ 5 DEG C washing with alcohol dryings.
First being organized in homogenate of middle remote tree shrew is carried out centrifugal extraction Mitochondrial DNA by the present invention, the mass volume ratio of middle remote tree shrew tissue and homogenate is preferably 0.2g:1mL, described centrifugal condition optimization is 4 DEG C, 1500r/min, and centrifugation time is preferably 10min.Described homogenate comprises sucrose, Na
2eDTA, Tris-HCl, CaCl
2, preferably include 0.25mol/L sucrose, 10mmol/L Na
2eDTA, 30mmol/L Tris-HCl and 2.5mmol/L CaCl
2.The pH value of described homogenate is preferably 8.2.
After first time centrifugal acquisition supernatant liquor, continue at 3 ~ 5 DEG C, centrifugal 18min ~ 22min under 1200r/min ~ 1800r/min condition.The centrifugal condition optimization of second time is 4 DEG C, the centrifugal 20min of 1500r/min.
After second time is centrifugal, what obtain after abandoning supernatant liquor is deposited in homogenate at 3 ~ 5 DEG C, centrifugal 8min ~ 12min under 1200r/min ~ 1800r/min condition.Third time, centrifugal condition optimization was 4 DEG C, the centrifugal 10min of 1500r/min.The homogenate of third time centrifugal use is identical with the homogenate of first time centrifugal upper use, comprises sucrose, Na
2eDTA, Tris-HCl, CaCl
2, preferably include 0.25mol/L sucrose, 10mmol/L Na
2eDTA, 30mmol/L Tris-HCl and 2.5mmol/L CaCl
2.The pH value of described homogenate is preferably 8.2.The homogenate of third time centrifugal use is 1.5 ~ 2.5mL:0.15 ~ 0.25g with the volume mass ratio of middle remote tree shrew tissue, is preferably 2mL:0.2g.
After third time is centrifugal, the precipitation obtained is mixed with the first solution, the more molten 8min ~ 12min of ice after mixing with the second solution.Wherein, described first solution comprises Tris-HCl, Na
2eDTA and NaCl, preferably includes 10mmol/L Tris-HCl, 10mmol/L Na
2eDTA and 0.15mmol/L NaCl; The pH value of described first solution is 8.0.Blow and beat gently after the precipitation that obtains after centrifugal for third time is mixed with the first solution and mix, then mix the molten 8min ~ 12min of ice, the molten 10min of preferred ice afterwards with the second solution.Described second solution comprises NaOH and sodium lauryl sulphate, preferably includes sodium lauryl sulphate and the 0.2mmol/L NaOH of 1wt%.The volume mass ratio of described first solution, the second solution and middle remote tree shrew tissue is 40 ~ 60 μ L:90 ~ 110 μ L:0.15 ~ 0.25g, is more preferably 50 μ L:100 μ L:0.2g.
And then the solution obtained is mixed with the 3rd solution, the molten 50min ~ 70min of ice, preferred 60min.Described 3rd solution comprises potassium ion and acetic acid ion, and wherein, potassium concentration is preferably 3mol/L, and described acetate ion concentration is preferably 5mol/L.The volume ratio of the 3rd solution and the second solution is 70 ~ 80:90 ~ 110, is preferably 75:100.
By the solution centrifugal 8min ~ 12min under 11000r/min ~ 13000r/min condition obtained, the 4th time centrifugal condition optimization is 12000r/min, centrifugal 10min.
By the supernatant liquor of the 4th centrifugal acquisition in mixing solutions under 11000r/min ~ 13000r/min condition extracting 2 ~ 3min, repeat once, extraction conditions is preferably 12000r/min.Wherein, described mixing solutions is phenol chloroform, comprises phenol, chloroform and primary isoamyl alcohol, and the volume ratio of phenol, chloroform and primary isoamyl alcohol is 24:24:1.The volume ratio of described supernatant liquor and mixing solutions is 0.8 ~ 1.2:0.8 ~ 1.2, is preferably 1:1.
The dehydrated alcohol of the supernatant liquor obtained after extracting with 3 DEG C ~ 5 DEG C is mixed, at-35 DEG C ~-45 DEG C left overnight.Wherein, dehydrated alcohol is preferably 4 DEG C, places the temperature of spending the night and is preferably-40 DEG C.
At the solution of freeze overnight at 3 DEG C ~ 5 DEG C, centrifugal 15min ~ 30min under 11000r/min ~ 13000r/min, with 4 DEG C of washing with alcohol dryings, can extract and obtain DNA.Wherein, the 5th centrifugal condition optimization is 4 DEG C, 12000r/min.Described ethanol is preferably the ethanol of 75%.
An exemplary embodiments of extracting method provided by the invention is as follows:
A) middle remote tree shrew to be organized in homogenate 4 DEG C, centrifugal 10min under 1500r/min condition, the mass volume ratio of described middle remote tree shrew tissue and homogenate is 0.2g:1mL;
B) supernatant liquor step a) obtained is at 4 DEG C, centrifugal 20min under 1500r/min condition;
C) by step c) obtain be deposited at 4 DEG C, centrifugal 10min under 1500r/min condition in homogenate, the volume mass of described homogenate and middle remote tree shrew tissue is than being 2mL:0.2g;
D) by step c) precipitation that obtains mixes with the first solution, the more molten 10min of ice after mixing with the second solution; Described first solution comprises Tris-HCl, Na
2eDTA and NaCl; Described second solution comprises NaOH and sodium lauryl sulphate; The volume mass ratio of described first solution, the second solution and middle remote tree shrew tissue is 50 μ L:100 μ L:0.2g;
E) by steps d) solution that obtains mixes with the 3rd solution, the molten 60min of ice; Described 3rd solution comprises potassium ion and acetate ion; The volume ratio of described 3rd solution and the second solution is 75:100;
F) by step e) solution centrifugal 10min under 12000r/min condition of obtaining;
G) by step f) supernatant liquor that obtains in mixing solutions under 12000r/min condition extracting 2 ~ 3min, repeat once; Described mixing solutions comprises phenol, chloroform and primary isoamyl alcohol; The volume ratio of described supernatant liquor and mixing solutions is 1:1;
H) by step g) supernatant liquor that obtains mixes with the dehydrated alcohol of 4 DEG C, spends the night-40 DEG C of placements;
I) by step h) solution that obtains at 4 DEG C, centrifugal 15min ~ 30min under 12000r/min, with 4 DEG C of washing with alcohol dryings.
The present invention is by limiting reagent type, reagent dosage and extraction conditions in leaching process, and extract the DNA purity obtained higher, electrophoretic band is without conditions of streaking, and cost is lower.
Accompanying drawing explanation
Fig. 1 is the electrophorogram that the embodiment of the present invention extracts the DNA obtained, and wherein, 1 and 2 electrophoretic bands being respectively the DNA that embodiment 1 and embodiment 2 obtain, 3 is the electrophoretic band of the DNA that comparative example 1 obtains.
Embodiment
Below in conjunction with the subordinate list in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
A) tree shrew remote in 0.2g to be organized in 1mL homogenate 4 DEG C, centrifugal 10min under 1500r/min condition; Described homogenate comprises 0.25mol/L sucrose, 10mmol/L Na
2eDTA, 30mmol/L Tris-HCl and 2.5mmol/LCaCl
2;
B) supernatant liquor step a) obtained is at 4 DEG C, centrifugal 20min under 1500r/min condition;
C) by step c) being deposited at 4 DEG C, centrifugal 10min under 1500r/min condition in 2mL homogenate of obtaining, described homogenate comprises 0.25mol/L sucrose, 10mmol/L Na
2eDTA, 30mmol/L Tris-HCl and 2.5mmol/L CaCl
2;
D) by step c) precipitation that obtains mixes with 50 μ L first solution, the more molten 10min of ice after mixing with 100 μ L second solution; Described first solution comprises 10mmol/L Tris-HCl, 10mmol/L Na
2eDTA and 0.15mmol/L NaCl, pH value is 8.0; Described second solution comprises sodium lauryl sulphate (SDS) and the 0.2mmol/L NaOH of 1wt%;
E) by steps d) solution that obtains mixes with 75 μ L the 3rd solution, the molten 60min of ice; Described 3rd solution comprises potassium ion and acetate ion, and wherein, potassium concentration is preferably 3mol/L, and described acetate ion concentration is preferably 5mol/L;
F) by step e) solution centrifugal 10min under 12000r/min condition of obtaining;
G) by step f) supernatant liquor that obtains in equal-volume mixing solutions under 12000r/min condition extracting 2min, repeat once; Described mixing solutions comprises phenol, chloroform and the primary isoamyl alcohol that volume ratio is 24:24:1;
H) by step g) supernatant liquor that obtains mixes with the dehydrated alcohol of 4 DEG C, spends the night-40 DEG C of placements;
I) by step h) solution that obtains is at 4 DEG C, centrifugal 30min under 12000r/min, dry by the washing with alcohol of 4 DEG C 75%.
Embodiment 2
A) tree shrew remote in 0.2g to be organized in 1mL homogenate 4 DEG C, centrifugal 10min under 1500r/min condition; Described homogenate comprises 0.25mol/L sucrose, 10mmol/L Na
2eDTA, 30mmol/L Tris-HCl and 2.5mmol/LCaCl
2;
B) supernatant liquor step a) obtained is at 4 DEG C, centrifugal 20min under 1500r/min condition;
C) by step c) being deposited at 4 DEG C, centrifugal 10min under 1500r/min condition in 2mL homogenate of obtaining, described homogenate comprises 0.25mol/L sucrose, 10mmol/L Na
2eDTA, 30mmol/L Tris-HCl and 2.5mmol/L CaCl
2;
D) by step c) precipitation that obtains mixes with 50 μ L first solution, the more molten 10min of ice after mixing with 100 μ L second solution; Described first solution comprises 10mmol/L Tris-HCl, 10mmol/L Na
2eDTA and 0.15mmol/L NaCl, pH value is 8.0; Described second solution comprises sodium lauryl sulphate (SDS) and the 0.2mmol/L NaOH of 1wt%;
E) by steps d) solution that obtains mixes with 75 μ L the 3rd solution, the molten 60min of ice; Described 3rd solution comprises potassium ion and acetate ion, and wherein, potassium concentration is preferably 3mol/L, and described acetate ion concentration is preferably 5mol/L;
F) by step e) solution centrifugal 10min under 12000r/min condition of obtaining;
G) by step f) supernatant liquor that obtains in equal-volume mixing solutions under 12000r/min condition extracting 3min, repeat once; Described mixing solutions comprises phenol, chloroform and the primary isoamyl alcohol that volume ratio is 24:24:1;
H) by step g) supernatant liquor that obtains mixes with the dehydrated alcohol of 4 DEG C, spends the night-40 DEG C of placements;
I) by step h) solution that obtains is at 4 DEG C, centrifugal 15min under 12000r/min, dry by the washing with alcohol of 4 DEG C 75%.
Comparative example 1
The DNA that embodiment 1, embodiment 2 and comparative example 1 obtain is carried out electrophoresis, result is the electrophorogram that the embodiment of the present invention extracts the DNA obtained see Fig. 1, Fig. 1, wherein, 1 and 2 electrophoretic bands being respectively the DNA that embodiment 1 and embodiment 2 obtain, 3 is the electrophoretic band of the DNA that comparative example 1 obtains.As shown in Figure 1, the electrophoretic band that method provided by the invention obtains is without hangover, and purity is high.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (8)
1. an extracting method for remote tree shrew tissue DNA in, comprises the following steps:
A) be organized in by middle remote tree shrew at 3 ~ 5 DEG C, centrifugal 8min ~ 12min under 1200r/min ~ 1800r/min condition in homogenate, the mass volume ratio of described middle remote tree shrew tissue and homogenate is 0.15 ~ 0.25g:0.5 ~ 1.5mL;
B) supernatant liquor step a) obtained is at 3 ~ 5 DEG C, centrifugal 18min ~ 22min under 1200r/min ~ 1800r/min condition;
C) by step c) being deposited at 3 ~ 5 DEG C, centrifugal 8min ~ 12min under 1200r/min ~ 1800r/min condition in homogenate of obtaining, described homogenate is 1.5 ~ 2.5mL:0.15 ~ 0.25g with the volume mass ratio of middle remote tree shrew tissue;
D) by step c) precipitation that obtains mixes with the first solution, the more molten 8min ~ 12min of ice after mixing with the second solution; Described first solution comprises Tris-HCl, Na
2eDTA and NaCl; Described second solution comprises NaOH and sodium lauryl sulphate; The volume mass ratio of described first solution, the second solution and middle remote tree shrew tissue is 40 ~ 60 μ L:90 ~ 110 μ L:0.15 ~ 0.25g;
E) by steps d) solution that obtains mixes with the 3rd solution, the molten 50min ~ 70min of ice; Described 3rd solution comprises potassium ion and acetic acid ion; The volume ratio of described 3rd solution and the second solution is 70 ~ 80:90 ~ 110;
F) by step e) solution centrifugal 8min ~ 12min under 11000r/min ~ 13000r/min condition of obtaining;
G) by step f) supernatant liquor that obtains in mixing solutions under 11000r/min ~ 13000r/min condition extracting 2 ~ 3min, repeat once; Described mixing solutions comprises phenol, chloroform and primary isoamyl alcohol; The volume ratio of described supernatant liquor and described mixing solutions is 0.8 ~ 1.2:0.8 ~ 1.2;
H) by step g) supernatant liquor that obtains mixes with the dehydrated alcohol of 3 DEG C ~ 5 DEG C, spends the night-35 DEG C ~-45 DEG C placements;
I) by step h) solution that obtains at 3 DEG C ~ 5 DEG C, centrifugal 15min ~ 30min under 11000r/min ~ 13000r/min, with 3 DEG C ~ 5 DEG C washing with alcohol dryings.
2. extracting method according to claim 1, is characterized in that, comprising:
A) middle remote tree shrew to be organized in homogenate 4 DEG C, centrifugal 10min under 1500r/min condition, the mass volume ratio of described middle remote tree shrew tissue and homogenate is 0.2g:1mL;
B) supernatant liquor step a) obtained is at 4 DEG C, centrifugal 20min under 1500r/min condition;
C) by step c) obtain be deposited at 4 DEG C, centrifugal 10min under 1500r/min condition in homogenate, the volume mass of described homogenate and middle remote tree shrew tissue is than being 2mL:0.2g;
D) by step c) precipitation that obtains mixes with the first solution, the more molten 10min of ice after mixing with the second solution; Described first solution comprises Tris-HCl, Na
2eDTA and NaCl; Described second solution comprises NaOH and sodium lauryl sulphate; The volume mass ratio of described first solution, the second solution and middle remote tree shrew tissue is 50 μ L:100 μ L:0.2g;
E) by steps d) solution that obtains mixes with the 3rd solution, the molten 60min of ice; Described 3rd solution comprises potassium ion and acetate ion; The volume ratio of described 3rd solution and the second solution is 75:100;
F) by step e) solution centrifugal 10min under 12000r/min condition of obtaining;
G) by step f) supernatant liquor that obtains in mixing solutions under 12000r/min condition extracting 2 ~ 3min, repeat once; Described mixing solutions comprises phenol, chloroform and primary isoamyl alcohol; The volume ratio of described supernatant liquor and mixing solutions is 1:1;
H) by step g) supernatant liquor that obtains mixes with the dehydrated alcohol of 4 DEG C, spends the night-40 DEG C of placements;
I) by step h) solution that obtains at 4 DEG C, centrifugal 15min ~ 30min under 12000r/min, with 4 DEG C of washing with alcohol dryings.
3. extracting method according to claim 1 and 2, is characterized in that, described homogenate comprises: 0.25mol/L sucrose, 10mmol/L Na
2eDTA, 30mmol/L Tris-Hcl, 2.5mmol/L CaCl
2.
4. extracting method according to claim 1 and 2, is characterized in that, described first solution comprises: 10mmol/L Tris-HCl, 10mmol/L Na
2eDTA and 0.15mmol/LNaCl.
5. extracting method according to claim 1 and 2, is characterized in that, described second solution comprises: the sodium lauryl sulphate of 1wt% and 0.2mmol/L NaOH.
6. extracting method according to claim 1 and 2, is characterized in that, in described 3rd solution, potassium concentration is 3mol/L, and described acetate ion concentration is 5mol/L.
7. extracting method according to claim 1 and 2, is characterized in that, in described mixing solutions, the volume ratio of phenol, chloroform and primary isoamyl alcohol is 24:24:1.
8. extracting method according to claim 1 and 2, is characterized in that, described step I) in, described ethanol is 75% ethanol.
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