CN102140448A - Method for extracting DNA from animal tissue - Google Patents

Abstract

The invention discloses a method for extracting DNA from animal tissue, which is easy to operate and high in extraction speed and ensures the extracted DNA is high in purity. The method comprises the following steps of: taking 50mg of animal tissue, cutting into pieces, putting into a homogenizer, and adding 300mu l of homogenizing liquid for grinding in an ice bath; adding 3mu l of 10mg/ml protease K and 15mu l of 10 percent sodium dodecyl sulfonate, uniformly mixing, and putting into a water bath at 55 DEG C for 2 to 4 hours until the solution is transparent and thick; adding 5M potassium acetate until the final concentration is 1M, uniformly mixing, and standing at 4 DEG C for 30 minutes; centrifuging at 4 DEG C for 10 minutes at a speed of 12,000r/min; taking supernate, transferring into another centrifuge tube, adding pre-cooled absolute ethanol in a volume which is two times that of the supernate, and standing at 4 DEG C for 1 hour; centrifuging at 4 DEG C for 10 minutes at a speed of 12,000r/min; discarding the supernate, washing and precipitating twice by using pre-cooled 70 percent ethanol, drying at room temperature, and dissolving by using 50mu l of tris-EDTA(ethylene diamine tetraacetic acid) (TE); and adding 10mg/ml RnaseA in a volume which is 1/50 of the volume of the solution in the step e, and keeping constant temperature of 37 DEG C for 1 hour.

Description

Animal tissues's DNA extraction method

Technical field:

The present invention relates to a kind of animal tissues DNA extraction method, especially a kind of simple to operate, not only extraction rate is fast, and the high animal tissues's DNA extraction method of the DNA purity of being mentioned.

Background technology:

Nucleic acid and the protein form with nucleoprotein in organism exists, and wherein DNA mainly is present in the nucleus, and RNA mainly is present in kernel and the kytoplasm.Usually when preparation nucleic acid, should prevent peracid, cross alkali and other can cause the factor effect of nucleolysis, should under low temperature (4 ℃), carry out as whole operating process, to add citric acid, prussiate, nitrate, ethylenediamine tetraacetic acid (EDTA) enzyme inhibitorss such as (EDTA) in case of necessity, also will avoid protein denaturants such as SDS or phenol etc. to make the nucleolysis enzyme destroyed simultaneously.The method that tradition is extracted DNA is earlier DNA and RNA branch to be opened, and DNA is separated with protein again.Action principle is because animals and plants DNA nucleoprotein energy is water-soluble and the salts solution (as: 1mol/L NaCl) of high density, but solubleness is very low in the salts solution of 0.14mol/L, RNA nucleoprotein then is dissolved in the 0.14mol/L salts solution, therefore can utilize different concentrations of sodium chloride solution, deoxyribonucleoprotein and ribonucleoprotein are detached out respectively from sample.Again deoxyribonucleoprotein SDS (sodium lauryl sulphate) is handled, DNA promptly separates with protein, and available chloroform-primary isoamyl alcohol is removed protein precipitation, DNA then dissolve with solution in, add cold ethanol in the water that contains DNA, DNA promptly is fibrous being precipitated out.Leaching process material therefor, reagent are more, complicated operation and consuming time longer.Particularly remove proteinic operation, if concussion acutely can make dna break, cause and extract the content reduction, can shake inadequately, then protein can not finely be removed, and the DNA that is extracted is impure.

Summary of the invention:

The present invention is in order to solve the above-mentioned technical problem of existing in prior technology, provide a kind of simple to operate, not only extraction rate is fast, and the high animal tissues's DNA extraction method of the DNA purity of being mentioned.

Technical solution of the present invention is: a kind of animal tissues DNA extraction method is characterized in that carrying out as follows:

A. get the 50mg of animal tissues, shred and be placed in the homogenizer, add 300 μ l homogenate ice baths and grind, described homogenate is 0.06M EDTA, 0.1M Tris-HCl, 0.16M sucrose, 0.08M NaCl;

B. add 3 μ l 10mg/mlde Proteinase Ks, 15 μ l 10%SDS, be transparent thick in mid-2～4 hours of 55 ℃ of water-baths to solution behind the mixing;

C. add the 5M potassium acetate to final concentration 1M, mixing is placed 30min for 4 ℃;

D.4 ℃, the centrifugal 10min of 12000r/min; Get supernatant liquor and move in another centrifuge tube, add the dehydrated alcohol of the long-pending precooling of diploid, place 1h for 4 ℃;

E.4 ℃, the centrifugal 10min of 12000r/min; Abandon supernatant, drying at room temperature after 70% washing with alcohol of usefulness precooling precipitates 2 times, 50 μ l TE dissolving;

F. the RnaseA that in e step gained solution, adds the 10mg/ml of 1/50 volume, 37 ℃ of constant temperature 1h.

The present invention removes the protein in the nucleoprotein with proteolytic enzyme earlier, with ethanol nucleic acid is slightly purified again, digests RNA with RnaseA more at last, and then DNA and RNA branch are opened, and simple to operate, not only extraction rate is fast, and the DNA purity height of being mentioned

Description of drawings:

Fig. 1 is the electrophoretogram of DNA that the embodiment of the invention is extracted.

Embodiment:

A. get fish liver organization 50mg, shred and be placed in the homogenizer, add 300 μ l homogenate ice baths and grind, described homogenate is 0.06M EDTA, 0.1M Tris-HCl, 0.16M sucrose, 0.08M NaCl;

B. add 3 μ l10mg/mlde Proteinase Ks, 15 μ l10%SDS, be transparent thick in mid-2～4 hours of 55 ℃ of water-baths to solution behind the mixing;

C. add the 5M potassium acetate to final concentration 1M, mixing is placed 30min for 4 ℃;

D.4 ℃, the centrifugal 10min of 12000r/min; Get supernatant liquor and move in another centrifuge tube, add the dehydrated alcohol of the long-pending precooling of diploid, place 1h for 4 ℃;

E.4 ℃, the centrifugal 10min of 12000r/min; Abandon supernatant, drying at room temperature after 70% washing with alcohol of usefulness precooling precipitates 2 times, 50 μ l TE dissolving;

F. the RnaseA that in e step gained solution, adds the 10mg/ml of 1/50 volume, 37 ℃ of constant temperature 1h.

Action principle:

Add enzyme inhibitors in the preparation process,, prevent nucleolysis as ethylenediamine tetraacetic acid (EDTA) (EDTA);

The nucleoprotein protease treatment can be removed protein wherein; Aqueous phase to nucleic acid adds cold ethanol, and nucleic acid promptly is fibrous being precipitated out.

RnaseA can digest RNA and can not dna digestion, so remove RNA in adjusting with RnaseA digestion.

Electrophoretic analysis: get the DNA sample 2 μ l that extracted and mix with 2 μ l tetrabromophenol sulfonphthaleins, HindIII DNA marker makes standard molecular weight, and 1% agarose gel electrophoresis (containing 0.5 μ g/ml EB) detects its integrity, and voltage is 80V, electrophoresis 40 minutes.Take pictures with gel imaging system, the molecular weight of sample estimates DNA and the concentration of DNA sample according to a preliminary estimate, as shown in Figure 1: complete DNA presents a high brightness master tape clearly when electrophoresis.

UV spectrophotometer measuring: get the DNA sample 10 μ l that extracted and be diluted to 2ml, on ultraviolet spectrophotometer, survey A ₂₆₀And A ₂₈₀The OD value, its OD ₂₆₀/ OD ₂₈₀Value should be about 1.8.

Claims (1)

1. animal tissues's DNA extraction method is characterized in that carrying out as follows:

A. get the 50mg of animal tissues, shred and be placed in the homogenizer, add 300 μ l homogenate ice baths and grind, described homogenate is 0.06M EDTA, 0.1M Tris-HCl, 0.16M sucrose, 0.08M NaCl;

B. add 3 μ l 10mg/mlde Proteinase Ks, 15 μ l10%SDS, be transparent thick in mid-2～4 hours of 55 ℃ of water-baths to solution behind the mixing;

C. add the 5M potassium acetate to final concentration 1M, mixing is placed 30min for 4 ℃;

D.4 ℃, the centrifugal 10min of 12000r/min; Get supernatant liquor and move in another centrifuge tube, add the dehydrated alcohol of the long-pending precooling of diploid, place 1h for 4 ℃;

E.4 ℃, the centrifugal 10min of 12000r/min; Abandon supernatant, drying at room temperature after 70% washing with alcohol of usefulness precooling precipitates 2 times, 50 μ lTE dissolving;

F. the RnaseA that in e step gained solution, adds the 10mg/ml of 1/50 volume, 37 ℃ of constant temperature 1h.

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