CN104830836B - A kind of extracting method of middle remote tree shrew tissue DNA - Google Patents
A kind of extracting method of middle remote tree shrew tissue DNA Download PDFInfo
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Abstract
The invention provides a kind of extracting method of middle remote tree shrew tissue DNA, comprise the following steps：A) by middle remote tree shrew be organized in homogenate from；B) supernatant for obtaining step a) centrifuges；C) being deposited in homogenate for step c) acquisitions is centrifuged；D) the step c) precipitations obtained are well mixed with the first solution, then ice is molten after being mixed with the second solution；E) the step d) solution obtained is mixed with the 3rd solution, ice is molten；F) solution centrifugal for obtaining step e)；G) the step f) supernatants obtained are extracted in mixed solution, be repeated once；H) the step g) supernatants obtained are mixed with absolute ethyl alcohol, stood overnight；I) solution centrifugal for obtaining step h), drying is washed with ethanol.The present invention is by limiting reagent type in extraction process, reagent dosage and extraction conditions, and the DNA purity for extracting to obtain is higher, and electrophoretic band is without conditions of streaking, and cost is relatively low.
Description
Technical field
The invention belongs to DNA extractive techniques field, more particularly to a kind of extracting method of middle remote tree shrew tissue DNA.
Background technology
Middle remote tree shrew be it is a kind of it is small-sized climb up by holding on to type mammal, be similar to squirrel, fastened in kind conventional compared with mouse, rat etc.
Laboratory rodent can suffer from many diseases similar to people closer to the mankind, be at present except chimpanzee, gibbon
Outside, the animal of human hepatitis B virus can be uniquely infected, receives biology and the highest attention of medical worker.
When centering Burma tree shrew is studied, it is one of essential step to extract its tissue DNA.Prior art discloses
The extracting method of other many animal DNAs, but these methods are not suitable for middle remote tree shrew tissue NDA extraction, and extraction obtains
DNA purity it is relatively low, DNA electrophoretic band conditions of streaking is serious.
The content of the invention
It is an object of the invention to provide a kind of extracting method of middle remote tree shrew tissue DNA, extraction side provided by the invention
The DNA purity that method extracts to obtain middle remote tree shrew is higher, 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) middle remote tree shrew is organized in homogenate and centrifuges 8min under the conditions of 3~5 DEG C, 1200r/min~1800r/min
~12min, the mass volume ratio of the middle remote tree shrew tissue and homogenate is 0.15~0.25g：0.5~1.5mL；
B) by step a) obtain supernatant centrifuged under the conditions of 3~5 DEG C, 1200r/min~1800r/min 18min~
22min；
C) being deposited in homogenate for step c) acquisitions is centrifuged under the conditions of 3~5 DEG C, 1200r/min~1800r/min
8min~12min, the volume mass ratio of the homogenate and middle remote tree shrew tissue is 1.5~2.5mL：0.15~0.25g；
D) the step c) precipitations obtained are well mixed with the first solution, then the molten 8min of ice after being mixed with the second solution~
12min；First solution includes Tris-HCl, Na2EDTA and NaCl；Second solution includes NaOH and dodecyl sulphur
Sour sodium；The volume mass ratio of 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) the step d) solution obtained is mixed with the 3rd solution, the molten 50min~70min of ice；3rd solution includes
Potassium ion and acetic acid ion；The volume ratio of 3rd solution and the second solution is 70~80:90~110；
F) the step e) solution obtained is centrifuged into 8min~12min under the conditions of 11000r/min~13000r/min；
G) the step f) supernatants obtained are extracted 2 in mixed solution under the conditions of 11000r/min~13000r/min
~3min, is repeated once；The mixed solution includes phenol, chloroform and isoamyl alcohol；The body of the supernatant and the mixed solution
Product is than being 0.8~1.2:0.8~1.2；
H) the step g) supernatants obtained are mixed with 3 DEG C~5 DEG C of absolute ethyl alcohol, placed at -35 DEG C~-45 DEG C
Night；
I) by step h) obtain solution centrifuged under 3 DEG C~5 DEG C, 11000r/min~13000r/min 15min~
30min, drying is washed with 3 DEG C~5 DEG C ethanol.
Being organized in homogenate for middle remote tree shrew is carried out centrifugation extraction mitochondrial DNA, middle remote tree shrew group by the present invention first
It is preferably 0.2g to knit with the mass volume ratio of homogenate：1mL, the condition of the centrifugation are preferably 4 DEG C, 1500r/min, during centrifugation
Between be preferably 10min.The homogenate includes sucrose, Na2EDTA、Tris-HCl、CaCl2, preferably include 0.25mol/L sugarcanes
Sugar, 10mmol/L Na2EDTA, 30mmol/L Tris-HCl and 2.5mmol/L CaCl2.The pH value of the homogenate is preferably
8.2。
By first time centrifuge obtain supernatant after, continue under the conditions of 3~5 DEG C, 1200r/min~1800r/min from
Heart 18min~22min.The condition of second of centrifugation is preferably 4 DEG C, 1500r/min centrifugations 20min.
After second centrifuges, abandon after supernatant being deposited in homogenate of obtaining 3~5 DEG C, 1200r/min~
8min~12min is centrifuged under the conditions of 1800r/min.The condition of third time centrifugation is preferably 4 DEG C, 1500r/min centrifugations 10min.
It is identical with the homogenate used in first time centrifugation to centrifuge the homogenate used for the third time, including sucrose, Na2EDTA、Tris-
HCl、CaCl2, preferably include 0.25mol/L sucrose, 10mmol/L Na2EDTA, 30mmol/L Tris-HCl and 2.5mmol/L
CaCl2.The pH value of the homogenate is preferably 8.2.The volume matter of the homogenate and middle remote tree shrew tissue that use is centrifuged for the third time
It is 1.5~2.5mL to measure ratio：0.15~0.25g, preferably 2mL：0.2g.
After third time centrifuges, obtained precipitation is well mixed with the first solution, then ice after being mixed with the second solution
Molten 8min~12min.Wherein, first solution includes Tris-HCl, Na2EDTA and NaCl, preferably includes 10mmol/L
Tris-HCl、10mmol/L Na2EDTA and 0.15mmol/L NaCl；The pH value of first solution is 8.0.Will third time from
The precipitation obtained after the heart is gently blown and beaten after being mixed with the first solution and is well mixed, and ice is molten after then being mixed with the second solution
The molten 10min of 8min~12min, preferably ice.Second solution includes NaOH and lauryl sodium sulfate, preferably includes 1wt%
Lauryl sodium sulfate and 0.2mmol/L NaOH.The volume matter of first solution, the second solution and middle remote tree shrew tissue
It is 40~60 μ L to measure ratio：90~110 μ L：0.15~0.25g, more preferably 50 μ L：100μL：0.2g.
Then obtained solution is mixed with the 3rd solution again, ice molten 50min~70min, preferably 60min.Described 3rd
Solution includes potassium ion and acetic acid ion, wherein, potassium concentration is preferably 3mol/L, and the acetate ion concentration is preferably
5mol/L.The volume ratio of 3rd solution and the second solution is 70~80:90~110, preferably 75:100.
The solution of acquisition is centrifuged into 8min~12min, the 4th centrifugation under the conditions of 11000r/min~13000r/min
Condition be preferably 12000r/min, centrifuge 10min.
The supernatant that 4th centrifugation is obtained is taken out in mixed solution under the conditions of 11000r/min~13000r/min
2~3min is carried, is repeated once, extraction conditions are preferably 12000r/min.Wherein, the mixed solution is phenol chloroform, including phenol,
Chloroform and isoamyl alcohol, the volume ratio of phenol, chloroform and isoamyl alcohol is 24:24:1.The volume ratio of the supernatant and mixed solution is
0.8~1.2:0.8~1.2, preferably 1:1.
The supernatant obtained after extracting is mixed with 3 DEG C~5 DEG C of absolute ethyl alcohol, placed at -35 DEG C~-45 DEG C
Night.Wherein, absolute ethyl alcohol is preferably 4 DEG C, and the temperature stood overnight is preferably -40 DEG C.
15min~30min is centrifuged under 3 DEG C~5 DEG C, 11000r/min~13000r/min in the solution of freeze overnight,
Drying is washed with 4 DEG C of ethanol, you can extraction obtains DNA.Wherein, the condition of the 5th centrifugation is preferably 4 DEG C, 12000r/min.
The ethanol is preferably 75% ethanol.
One exemplary embodiments of extracting method provided by the invention are as follows：
A) middle remote tree shrew is organized in homogenate 4 DEG C, centrifuges 10min, the middle remote tree shrew group under the conditions of 1500r/min
It is 0.2g to knit with the mass volume ratio of homogenate：1mL；
B) the step a) supernatants obtained are centrifuged into 20min under the conditions of 4 DEG C, 1500r/min；
C) being deposited in homogenate under the conditions of 4 DEG C, 1500r/min for step c) acquisitions is centrifuged into 10min, the homogenate
The volume mass ratio of liquid and middle remote tree shrew tissue is 2mL：0.2g；
D) the step c) precipitations obtained are well mixed with the first solution, then the molten 10min of ice after being mixed with the second solution；Institute
Stating the first solution includes Tris-HCl, Na2EDTA and NaCl；Second solution includes NaOH and lauryl sodium sulfate；Institute
The volume mass ratio for stating the first solution, the second solution and middle remote tree shrew tissue is 50 μ L：100μL：0.2g；
E) the step d) solution obtained is mixed with the 3rd solution, the molten 60min of ice；3rd solution include potassium ion and
Acetate ion；The volume ratio of 3rd solution and the second solution is 75:100；
F) the step e) solution obtained is centrifuged into 10min under the conditions of 12000r/min；
G) supernatant for obtaining step f) extracts 2~3min in mixed solution under the conditions of 12000r/min, repeats
Once；The mixed solution includes phenol, chloroform and isoamyl alcohol；The volume ratio of the supernatant and mixed solution is 1:1；
H) the step g) supernatants obtained are mixed with 4 DEG C of absolute ethyl alcohol, stood overnight at -40 DEG C；
I) the step h) solution obtained is centrifuged into 15min~30min under 4 DEG C, 12000r/min, washed with 4 DEG C of ethanol
Dry.
For the present invention by limiting reagent type in extraction process, reagent dosage and extraction conditions, the DNA for extracting to obtain is pure
Degree is higher, and electrophoretic band is without conditions of streaking, and cost is relatively low.
Brief description of the drawings
The electrophoretogram for the DNA that Fig. 1 extracts to obtain for the embodiment of the present invention, wherein, 1 and 2 be respectively embodiment 1 and embodiment
2 obtained DNA electrophoretic band, 3 be the electrophoretic band for the DNA that comparative example 1 obtains.
Embodiment
Below in conjunction with the subordinate list in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, rather than whole embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other under the premise of creative work is not made
Embodiment, belong to the scope of protection of the invention.
Embodiment 1
A) remote tree shrew in 0.2g is organized in 1mL homogenates 4 DEG C, centrifuges 10min under the conditions of 1500r/min；It is described even
Slurries include 0.25mol/L sucrose, 10mmol/L Na2EDTA, 30mmol/L Tris-HCl and 2.5mmol/L CaCl2；
B) the step a) supernatants obtained are centrifuged into 20min under the conditions of 4 DEG C, 1500r/min；
C) being deposited in 2mL homogenates for step c) acquisitions is centrifuged into 10min under the conditions of 4 DEG C, 1500r/min, it is described
Homogenate includes 0.25mol/L sucrose, 10mmol/L Na2EDTA, 30mmol/L Tris-HCl and 2.5mmol/L CaCl2；
D) the step c) precipitations obtained are well mixed with 50 the first solution of μ L, then ice after being mixed with 100 the second solution of μ L
Molten 10min；First solution includes 10mmol/L Tris-HCl, 10mmol/L Na2EDTA and 0.15mmol/L NaCl,
PH value is 8.0；Second solution includes 1wt% lauryl sodium sulfate (SDS) and 0.2mmol/L NaOH；
E) the step d) solution obtained is mixed with the solution of 75 μ L the 3rd, the molten 60min of ice；3rd solution include potassium from
Son and acetate ion, wherein, potassium concentration is preferably 3mol/L, and the acetate ion concentration is preferably 5mol/L；
F) the step e) solution obtained is centrifuged into 10min under the conditions of 12000r/min；
G) supernatant for obtaining step f) extracts 2min in isometric mixed solution under the conditions of 12000r/min, weight
Again once；It is 24 that the mixed solution, which includes volume ratio,:24:1 phenol, chloroform and isoamyl alcohol；
H) the step g) supernatants obtained are mixed with 4 DEG C of absolute ethyl alcohol, stood overnight at -40 DEG C；
I) the step h) solution obtained is centrifuged into 30min under 4 DEG C, 12000r/min, washed with 4 DEG C 75% of ethanol dry
It is dry.
Embodiment 2
A) remote tree shrew in 0.2g is organized in 1mL homogenates 4 DEG C, centrifuges 10min under the conditions of 1500r/min；It is described even
Slurries include 0.25mol/L sucrose, 10mmol/L Na2EDTA, 30mmol/L Tris-HCl and 2.5mmol/L CaCl2；
B) the step a) supernatants obtained are centrifuged into 20min under the conditions of 4 DEG C, 1500r/min；
C) being deposited in 2mL homogenates for step c) acquisitions is centrifuged into 10min under the conditions of 4 DEG C, 1500r/min, it is described
Homogenate includes 0.25mol/L sucrose, 10mmol/L Na2EDTA, 30mmol/L Tris-HCl and 2.5mmol/L CaCl2；
D) the step c) precipitations obtained are well mixed with 50 the first solution of μ L, then ice after being mixed with 100 the second solution of μ L
Molten 10min；First solution includes 10mmol/L Tris-HCl, 10mmol/L Na2EDTA and 0.15mmol/L NaCl,
PH value is 8.0；Second solution includes 1wt% lauryl sodium sulfate (SDS) and 0.2mmol/L NaOH；
E) the step d) solution obtained is mixed with the solution of 75 μ L the 3rd, the molten 60min of ice；3rd solution include potassium from
Son and acetate ion, wherein, potassium concentration is preferably 3mol/L, and the acetate ion concentration is preferably 5mol/L；
F) the step e) solution obtained is centrifuged into 10min under the conditions of 12000r/min；
G) supernatant for obtaining step f) extracts 3min in isometric mixed solution under the conditions of 12000r/min, weight
Again once；It is 24 that the mixed solution, which includes volume ratio,:24:1 phenol, chloroform and isoamyl alcohol；
H) the step g) supernatants obtained are mixed with 4 DEG C of absolute ethyl alcohol, stood overnight at -40 DEG C；
I) the step h) solution obtained is centrifuged into 15min under 4 DEG C, 12000r/min, washed with 4 DEG C 75% of ethanol dry
It is dry.
Comparative example 1
The DNA that embodiment 1, embodiment 2 and comparative example 1 are obtained carries out electrophoresis, as a result real for the present invention referring to Fig. 1, Fig. 1
The DNA electrophoretogram that example is extracted to obtain is applied, wherein, 1 and 2 be respectively the electrophoretic band for the DNA that embodiment 1 and embodiment 2 obtain,
3 be the electrophoretic band for the DNA that comparative example 1 obtains.As shown in Figure 1, the electrophoretic band that method provided by the invention obtains without hangover,
Purity is high.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (7)
1. a kind of extracting method of middle remote tree shrew tissue DNA, comprises the following steps：
A) by middle remote tree shrew be organized in homogenate under the conditions of 3~5 DEG C, 1200r/min~1800r/min centrifuge 8min~
12min, the mass volume ratio of the middle remote tree shrew tissue and homogenate is 0.15~0.25g：0.5~1.5mL；
B) by step a) obtain supernatant centrifuged under the conditions of 3~5 DEG C, 1200r/min~1800r/min 18min~
22min；
C) being deposited in homogenate for step c) acquisitions is centrifuged into 8min under the conditions of 3~5 DEG C, 1200r/min~1800r/min
~12min, the volume mass ratio of the homogenate and middle remote tree shrew tissue is 1.5~2.5mL：0.15~0.25g；
D) the step c) precipitations obtained are well mixed with the first solution, then the molten 8min~12min of ice after being mixed with the second solution；
First solution includes Tris-HCl, Na2EDTA and NaCl；Second solution includes NaOH and lauryl sodium sulfate；
The volume mass ratio of 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) the step d) solution obtained is mixed with the 3rd solution, the molten 50min~70min of ice；3rd solution include potassium from
Son and acetic acid ion；The volume ratio of 3rd solution and the second solution is 70~80:90~110；
F) the step e) solution obtained is centrifuged into 8min~12min under the conditions of 11000r/min~13000r/min；
G) by step f) obtain supernatant in mixed solution under the conditions of 11000r/min~13000r/min extracting 2~
3min, it is repeated once；The mixed solution includes phenol, chloroform and isoamyl alcohol；The volume of the supernatant and the mixed solution
Than for 0.8~1.2:0.8~1.2；
H) the step g) supernatants obtained are mixed with 3 DEG C~5 DEG C of absolute ethyl alcohol, stood overnight at -35 DEG C~-45 DEG C；
I) the step h) solution obtained is centrifuged into 15min~30min under 3 DEG C~5 DEG C, 11000r/min~13000r/min,
Drying is washed with 3 DEG C~5 DEG C ethanol；
Wherein, second solution includes：1wt% lauryl sodium sulfate and 0.2mmol/L NaOH.
2. extracting method according to claim 1, it is characterised in that including：
A) middle remote tree shrew is organized in homogenate 4 DEG C, 10min is centrifuged under the conditions of 1500r/min, the middle remote tree shrew tissue with
The mass volume ratio of homogenate is 0.2g：1mL；
B) the step a) supernatants obtained are centrifuged into 20min under the conditions of 4 DEG C, 1500r/min；
C) be deposited in homogenate under the conditions of 4 DEG C, 1500r/min by what step c) was obtained and centrifuge 10min, the homogenate with
The volume mass ratio of middle remote tree shrew tissue is 2mL：0.2g；
D) the step c) precipitations obtained are well mixed with the first solution, then the molten 10min of ice after being mixed with the second solution；Described
One solution includes Tris-HCl, Na2EDTA and NaCl；Second solution includes NaOH and lauryl sodium sulfate；Described
The volume mass ratio of one solution, the second solution and middle remote tree shrew tissue is 50 μ L：100μL：0.2g；
E) the step d) solution obtained is mixed with the 3rd solution, the molten 60min of ice；3rd solution includes potassium ion and acetic acid
Radical ion；The volume ratio of 3rd solution and the second solution is 75:100；
F) the step e) solution obtained is centrifuged into 10min under the conditions of 12000r/min；
G) supernatant for obtaining step f) extracts 2~3min in mixed solution under the conditions of 12000r/min, is repeated once；
The mixed solution includes phenol, chloroform and isoamyl alcohol；The volume ratio of the supernatant and mixed solution is 1:1；
H) the step g) supernatants obtained are mixed with 4 DEG C of absolute ethyl alcohol, stood overnight at -40 DEG C；
I) the step h) solution obtained is centrifuged into 15min~30min under 4 DEG C, 12000r/min, drying is washed with 4 DEG C of ethanol.
3. extracting method according to claim 1 or 2, it is characterised in that the homogenate includes：0.25mol/L sucrose,
10mmol/L Na2EDTA、30mmol/L Tris-Hcl、2.5mmol/L CaCl2。
4. extracting method according to claim 1 or 2, it is characterised in that first solution includes：10mmol/L
Tris-HCl、10mmol/L Na2EDTA and 0.15mmol/L NaCl.
5. extracting method according to claim 1 or 2, it is characterised in that in the 3rd solution, potassium concentration is
3mol/L, the acetate ion concentration are 5mol/L.
6. extracting method according to claim 1 or 2, it is characterised in that in the mixed solution, phenol, chloroform and isoamyl
The volume ratio of alcohol is 24:24:1.
7. extracting method according to claim 1 or 2, it is characterised in that in the step i), the ethanol is 75% second
Alcohol.
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| CN103911366A (en) * | 2014-03-19 | 2014-07-09 | 华南理工大学 | Genome DNA extraction method and its kit |
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