CN104152438A - Cell lysate used for extracting poultry DNA, and kit and method thereof - Google Patents
Cell lysate used for extracting poultry DNA, and kit and method thereof Download PDFInfo
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Abstract
The invention provides a cell lysate used for extracting poultry DNA. The cell lysate comprises 0.5-2.5g/L of Tris-HCl, 0.3-1.0g/L of KCl, 1.5-2.5g/L of MgCl2, 0.5-1.5g/L of EDTA, 20-30mL/L of Triton X-100, and the balance of water; and the pH value is adjusted by NaOH to 8. The invention also provides a poultry DNA extraction kit containing the cell lysate, and a poultry DNA extraction method. The bird blood DNA extraction time of the method is substantially shorter than that of traditional methods, the cost of the method is lower than that of a kit method when the extraction quality of the kit is reached or exceeded, and the method has few steps, and has lower operation difficulty than common kits.
Description
Technical field
The invention belongs to technical field of biological breeding, relate in particular to a kind of for extracting cell pyrolysis liquid, test kit and the method for poultry DNA.
Background technology
Poultry molecular breeding is as the indispensable technology of modern poultry breeding work, often need to carry out genetic background evaluation, genetic diseases rejecting, the purification of recessive inheritance proterties to core population, as recessive white feather equimolecular in chicken plumage look proterties detects related work to control progeny population quality.And DNA extraction is the basis of follow-up molecule experiment, therefore, extensive, quick obtaining poultry genome is very necessary to carrying out of poultry molecular breeding work.
For bird blood rbc have nuclear feature and for reduce poultry to sampling stress, mainly with venous blood collection under wing, be main at present, to reach, from a small amount of venous blood, extract enough DNA to meet the object of experiment needs.Prior art discloses multiple poultry blood DNA extractive technique, the Chinese patent literature that is 201210100069.2 as application number discloses a kind of method of extracting safely and fast genomic dna from blood, it is mixed into erythrocyte cracked liquid mutually with SDS (sodium lauryl sulphate) and Trito X-100 (polyoxyethylene glycol is to iso-octyl phenyl ether), saturated nacl aqueous solution is protein precipitant and uses ethanol precipitation DNA, the method extraction time is relatively short, can complete DNA and slightly extract work, can be widely used in blood DNA genome and extract.But the method obtains DNA and has higher protein contamination, and use sample size larger, can add resample burden, Animal stress, reduce blood sample access times, bird blood DNA is extracted to specific aim not strong, it is consuming time longer than the present invention.
Application number is that 201110030176.8 Chinese patent literature discloses a kind of cell pyrolysis liquid, test kit and the method that animal DNA extracts, take Guanidinium hydrochloride damping fluid as main lysate, and after adding Proteinase K, water-bath is spent the night, with saturated phenol extraction DNA, the method is further improved on traditional DNA extraction method phenol-chloroform extraction process, can obtain the DNA genome of certain mass.But the method length consuming time, needs digested overnight, extract whole process and reach more than 16 hours, be unfavorable for quick, batch operation.
Summary of the invention
In view of this, the object of the present invention is to provide a kind ofly for extracting cell pyrolysis liquid, test kit and the method for poultry DNA, that method provided by the invention is extracted is easy and simple to handle, the used time is short, and cost is low.
The invention provides a kind ofly for extracting the cell pyrolysis liquid of poultry DNA, comprising:
0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 20mL/L～30mL/L Triton X-100; The water of surplus; With NaOH, regulating pH value is 8.
As preferably, described cell pyrolysis liquid comprises:
1.576g/L Tris-HCl; 0.7455g/L KCl; 2.033g/L MgCl
2; 0.7448g/L EDTA; 25mL/L Triton X-100; The water of surplus; With NaOH, regulating pH value is 8.
It is a kind of for extracting the cell damping fluid of poultry DNA that the present invention also provides, and comprising:
0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 15g/L～30g/L NaCl; 0.5g/L～1.5g/L SDS; The water of surplus; With NaOH, regulating pH value is 8.
As preferably, described cell damping fluid comprises:
1.576g/L Tris-HCl; 0.7455g/L KCl; 2.033g/L MgCl
2; 0.7448g/L EDTA; 23.376g/L NaCl; 1g/L SDS; The water of surplus; With NaOH, regulating pH value is 8.
Above-mentioned cell pyrolysis liquid provided by the invention and cell damping fluid preferably combination are used, that is, it is a kind of for extracting the cell pyrolysis liquid of poultry DNA that the present invention also provides, and comprises Buffer WY and Buffer YW, wherein,
Described Buffer WY comprises: 0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 20mL/L～30mL/L Triton X-100; The water of surplus; With NaOH, regulating pH value is 8;
Described Buffer YW comprises: 0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 15g/L～30g/L NaCl; 0.5g/L～1.5g/L SDS; The water of surplus; With NaOH, regulating pH value is 8.
As preferably, described Buffer WY comprises: 1.576g/L Tris-HCl; 0.7455g/L KCl; 2.033g/L MgCl
2; 0.7448g/L EDTA; 25mL/L Triton X-100; The water of surplus; With NaOH, regulating pH value is 8;
Described Buffer YW comprises: 1.576g/L Tris-HCl; 0.7455g/L KCl; 2.033g/L MgCl
2; 0.7448g/L EDTA; 23.376g/L NaCl; 1g/L SDS; The water of surplus; With NaOH, regulating pH value is 8.
The present invention also provides a kind of poultry DNA extraction test kit that contains the cell pyrolysis liquid described in technique scheme, comprises Buffer WY and Buffer YW.
As preferably, described test kit also comprises: NaCl solution, Virahol, 75% alcohol and TE lysate.
As preferably, described test kit also comprises Proteinase K solution, and Proteinase K solution can make DNA purity and the receipts amount obtained higher.
As preferably, the concentration of described NaCl solution is 5mol/L.
As preferably, the concentration of described Proteinase K solution is 20mg/mL.
The present invention also provides a kind of poultry DNA extraction method, comprising:
A) fresh anti-freezing poultry blood sample is mixed with Buffer WY solution, centrifugal after heating in water bath, by abandoning the material obtaining after supernatant, mix with Buffer WY solution, abandon supernatant after centrifugal to be precipitated; Described Buffer WY solution comprises: 0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 20mL/L～30mL/L Triton X-100; The water of surplus; With NaOH, regulating pH value is 8;
B) precipitation described step a) being obtained is mixed with Buffer YW solution and Proteinase K solution, adds NaCl solution after heating in water bath, centrifugal after purifying in nucleic acid purification post, obtains filtrate; Described Buffer YW solution comprises: 0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 15g/L～30g/L NaCl; 0.5g/L～1.5g/L SDS; The water of surplus; With NaOH, regulating pH value is 8;
C) by described step b) filtrate that obtains mixes with Virahol, abandons supernatant after centrifugal, then mixes with 75% alcohol, abandons supernatant after centrifugal, adds TE lysate to dissolve after heating.
As preferably, described step a) is specially: the fresh anti-freezing poultry of 10～20 μ L blood sample is mixed with 1000 μ L Buffer WY solution concussions, centrifugal 1～the 3min of 10000～15000rpm/min after 60～70 ℃ of heating in water bath 5min～8min, by abandoning the material obtaining after supernatant, mix with 1000 μ L Buffer WY solution concussions, after the centrifugal 1～3min of 10000～15000rpm/min, abandon supernatant and be precipitated.
As preferably, described step is more specifically a): the fresh anti-freezing poultry of 10 μ L blood sample is mixed with 1000 μ L Buffer WY solution concussions, 60 ℃ of heating in water bath 8min, the centrifugal 2min of 12000rpm/min, by abandoning the material obtaining after supernatant, mix with 1000 μ L Buffer WY solution concussions, sedimentable matter is disperseed, after the centrifugal 2min of 12000rpm/min, abandon supernatant and be precipitated.
As preferably, described step b) be specially: the precipitation that described step a) is obtained is mixed with the Proteinase K solution concussion of 200 μ L Buffer YW solution and 10 μ L20mg/mL, after 60～70 ℃ of heating in water bath 20min～30min, add the NaCl solution of 100 μ L5mol/L, in nucleic acid purification post, the centrifugal 1min of 6000～10000rpm/min after purifying, obtains filtrate.
As preferably, described step b) be more specifically: the precipitation that described step a) is obtained is mixed with the Proteinase K solution concussion of 200 μ L Buffer YW solution and 10 μ L20mg/mL, and 60 ℃ of heating in water bath 25min also rock twice; Then the NaCl solution that adds 100 μ L5mol/L, after mixing in nucleic acid purification post purifying, the centrifugal 1min of 8000rpm/min, obtains filtrate, and filtrate is transferred in another 1.5mlEP pipe.
As preferably, described step c) being specially: by described step b) filtrate that obtains mixes with isopyknic Virahol, after the centrifugal 3～5min of 10000～15000rpm/min, abandon supernatant, then mix with 1000～1500 μ L75% alcohol vibrations, after the centrifugal 1～3min of 10000～15000rpm/min, abandon supernatant, after 70～75 ℃ of baking oven for heating 1～3min, add 50mLTE lysate to dissolve.
As preferably, described step c) being more specifically: by described step b) filtrate that obtains fully mixes with isopyknic Virahol, after the centrifugal 5min of 12000rpm/min, abandon supernatant, then mix with 1000 μ L75% alcohol vibrations, after mixing the centrifugal 3min of rear 12000rpm/min, abandon supernatant, after 72 ℃ of baking oven for heating 3min, add 50mLTE lysate to dissolve, sample is put in to-20 ℃ of Refrigerator stores standby.
As preferably, described bird blood sample can be goose blood, chicken blood, duck blood or pigeon blood.
In addition, method provided by the invention also can be without adding Proteinase K solution, and the DNA purity and the receipts amount that now obtain are lower slightly, but can meet regular-PCR requirement.
Compared with prior art, poultry DNA extraction method tool provided by the invention one of has the following advantages:
1, the invention solves current conventional DNA extraction method and need digest the problem of spending the night or needing the length consuming time such as a few hours digestion, significantly shorten ordinary method and extract bird blood sample DNA required time, by whole DNA extraction process control in 1 hour, the theoretical time is 49min, really realize the genomic object of quick obtaining poultry DNA, present method is extracted the test kit about 30min that saves time than TaKaRa whole blood genome.
2, the invention solves ordinary method and extract the too much problem of operation steps in DNA process, only need 6 steps can complete DNA extraction work, and TaKaRa whole blood genome extraction test kit needs 15 step operations, complex operation.
3, the present invention has overcome that test kit extraction cost is expensive, problem that cannot high volume applications: TaKaRa whole blood genome extracts approximately 10 yuan of samples of test kit cost, and sample cost of present method is 0.9 yuan of left and right.
4, the present invention makes whole DNA extraction process without drawing supernatant solution to the using method of nucleic acid purification post, sucting is clearly the rate-limiting step of DNA extraction, need careful operation, usually need to consume the plenty of time and easily cause protein contamination, manual operation factor is larger on result impact.The inventive method filters genomic dna with nucleic acid purification post, makes DNA in filtrate and the precipitations such as protein are stayed on filter membrane.The present invention is contrary with test kit to the using method of nucleic acid purification post, and test kit is to filter protein and DNA precipitation is stayed on filter membrane, without drawing supernatant liquor, uses nucleic acid purification post to filter DNA liquid and makes gained DNA protein contamination low, and purity is high, time saving and energy saving.
5, the present invention's extraction obtains DNA purity and receipts amount are higher, can meet or exceed test kit and extract quality.
In a word, method provided by the invention makes bird blood DNA extraction time than the remarkable shortening of traditional method, extracts the main flow test kit required times such as test kit shorter than TaKaRa whole blood genome.When meeting or exceeding test kit extraction quality, its cost is far below kit method, and step is few, and operation easier is lower than main flow test kit.The object that the present invention has realized is quick in laboratory, cheap, high quality obtains bird poba gene group DNA.
Accompanying drawing explanation
The DNA electrophoresis result that Fig. 1 extracts for the method that the embodiment of the present invention provides;
The DNA electrophoresis result that Fig. 2 extracts for the method that the embodiment of the present invention and comparative example provide;
Fig. 3 is the DNA profiling pcr amplification result that the embodiment of the present invention 1 is obtained;
The DNA electrophoresis result that Fig. 4 extracts for the method that the embodiment of the present invention 6～10 provides.
Embodiment
Below in conjunction with embodiment, to provided by the invention, for extracting cell pyrolysis liquid, test kit and the method for poultry DNA, be further described.
In following embodiment, agents useful for same is as follows: Buffer WY, Buffer YW, 5mol/L NaCl solution, Virahol, 75% alcohol, TE lysate, 20mg/ml Proteinase K solution;
Buffer WY:Tris-HCl1.576g, KCl0.7455g, MgCl
22.033g, EDTA0.7448g, Triton X-100 25ml, is settled to 1000ml, and NaOH regulates PH to 8.0.
Buffer YW:Tris-HCl1.576g, KCl0.7455g, MgCl
22.033g, EDTA0.7448g, NaCl23.376g, SDS1g, is settled to 1000ml, and NaOH regulates PH to 8.0.
Embodiment 1
1. get the fresh anti-freezing goose of 10ul blood in 1.5ml EP, add 1000ul Buffer WY solution, concussion mixes, and in 60 ℃ of water-bath 8min, after the centrifugal 2min of 12000rpm/min, abandons supernatant; In precipitation, add 1000ul BufferWY solution, concussion disperses precipitation, after the centrifugal 2min of 12000rpm/min, abandons supernatant again.
2. in precipitation obtained in the previous step, add 200ul Buffer YW solution and 20mg/ml Proteinase K solution 10ul, concussion mixes, in 60 ℃ of water-bath 25min and rock twice.
3. in previous step gained mixed solution, add 5mol/L NaCl solution 100ul, after mixing, complete soln is transferred in nucleic acid purification post, the centrifugal 1min of 8000rpm/min, proceeds to gained filtrate in the 1.5ml EP pipe renumbeing.
4. add equal-volume Virahol in above-mentioned filtrate, fully mix the centrifugal 5min of rear 12000rpm/min, abandon supernatant.
5. add 75% alcohol 1000ul, concussion mixes the centrifugal 3min of rear 12000rpm/min, abandons supernatant.
6. put into after 72 ℃ of baking oven 3min, add the TE lysate of 50ml to dissolve, sample is put in to-20 ℃ of Refrigerator stores standby.
7. gained DNA liquid carries out electrophoresis detection with 1.5% sepharose, and detects its concentration with nucleic acid-protein instrument NanoVue Plus, and result is referring to Fig. 1 and table 1, the DNA electrophoresis result that Fig. 1 extracts for the method that the embodiment of the present invention provides, and wherein, M is Marker; The nucleic acid-protein instrument detected result of the DNA that table 1 provides for the embodiment of the present invention and comparative example.As shown in Figure 1, the band of the DNA of method extraction goose blood provided by the invention is bright single.
Embodiment 2
1. get the fresh anti-freezing chicken of 10ul blood in 1.5ml EP, add 1000ul Buffer WY solution, concussion mixes, and in 60 ℃ of water-bath 8min, after the centrifugal 2min of 12000rpm/min, abandons supernatant; In precipitation, add 1000ul Buffer WY solution, concussion disperses precipitation, after the centrifugal 2min of 12000rpm/min, abandons supernatant again.
2. in precipitation obtained in the previous step, add 200ul Buffer YW solution and 20mg/ml Proteinase K solution 10ul, concussion mixes, in 60 ℃ of water-bath 25min and rock twice.
3. in previous step gained mixed solution, add 5mol/L NaCl solution 100ul, after mixing, complete soln is transferred in nucleic acid purification post, the centrifugal 1min of 8000rpm/min, proceeds to gained filtrate in the 1.5ml EP pipe renumbeing.
4. add equal-volume Virahol in above-mentioned filtrate, fully mix the centrifugal 5min of rear 12000rpm/min, abandon supernatant.
5. add 75% alcohol 1000ul, concussion mixes the centrifugal 3min of rear 12000rpm/min, abandons supernatant.
6. put into after 72 ℃ of baking oven 3min, add the TE lysate of 50ml to dissolve, sample is put in to-20 ℃ of Refrigerator stores standby.
7. gained DNA liquid carries out electrophoresis detection with 1.5% sepharose, and detects its concentration with nucleic acid-protein instrument NanoVue Plus, and result is referring to Fig. 1 and table 1, the DNA electrophoresis result that Fig. 1 extracts for the method that the embodiment of the present invention provides, and wherein, M is Marker; The nucleic acid-protein instrument detected result of the DNA that table 1 provides for the embodiment of the present invention and comparative example.As shown in Figure 1, the band of the DNA of method extraction chicken blood provided by the invention is bright single.
Embodiment 3
1. get the fresh anti-freezing pigeon of 10ul blood in 1.5ml EP, add 1000ul Buffer WY solution, concussion mixes, and in 60 ℃ of water-bath 8min, after the centrifugal 2min of 12000rpm/min, abandons supernatant; In precipitation, add 1000ul Buffer WY solution, concussion disperses precipitation, after the centrifugal 2min of 12000rpm/min, abandons supernatant again.
2. in precipitation obtained in the previous step, add 200ul Buffer YW solution and 20mg/ml Proteinase K solution 10ul, concussion mixes, in 60 ℃ of water-bath 25min and rock twice.
3. in previous step gained mixed solution, add 5mol/L NaCl solution 100ul, after mixing, complete soln is transferred in nucleic acid purification post, the centrifugal 1min of 8000rpm/min, proceeds to gained filtrate in the 1.5ml EP pipe renumbeing.
4. add equal-volume Virahol in above-mentioned filtrate, fully mix the centrifugal 5min of rear 12000rpm/min, abandon supernatant.
5. add 75% alcohol 1000ul, concussion mixes the centrifugal 3min of rear 12000rpm/min, abandons supernatant.
6. put into after 72 ℃ of baking oven 3min, add the TE lysate of 50ml to dissolve, sample is put in to-20 ℃ of Refrigerator stores standby.
7. gained DNA liquid carries out electrophoresis detection with 1.5% sepharose, and detects its concentration with nucleic acid-protein instrument NanoVue Plus, and result is referring to Fig. 1 and table 1, the DNA electrophoresis result that Fig. 1 extracts for the method that the embodiment of the present invention provides, and wherein, M is Marker; The nucleic acid-protein instrument detected result of the DNA that table 1 provides for the embodiment of the present invention and comparative example.As shown in Figure 1, the band of the DNA of method extraction pigeon blood provided by the invention is bright single.
Embodiment 4
1. get the fresh anti-freezing duck of 10ul blood in 1.5ml EP, add 1000ul Buffer WY solution, concussion mixes, and in 60 ℃ of water-bath 8min, after the centrifugal 2min of 12000rpm/min, abandons supernatant; In precipitation, add 1000ul Buffer WY solution, concussion disperses precipitation, after the centrifugal 2min of 12000rpm/min, abandons supernatant again.
2. in precipitation obtained in the previous step, add 200ul Buffer YW solution and 20mg/ml Proteinase K solution 10ul, concussion mixes, in 60 ℃ of water-bath 25min and rock twice.
3. in previous step gained mixed solution, add 5mol/L NaCl solution 100ul, after mixing, complete soln is transferred in nucleic acid purification post, the centrifugal 1min of 8000rpm/min, proceeds to gained filtrate in the 1.5ml EP pipe renumbeing.
4. add equal-volume Virahol in above-mentioned filtrate, fully mix the centrifugal 5min of rear 12000rpm/min, abandon supernatant.
5. add 75% alcohol 1000ul, concussion mixes the centrifugal 3min of rear 12000rpm/min, abandons supernatant.
6. put into after 72 ℃ of baking oven 3min, add the TE lysate of 50ml to dissolve, sample is put in to-20 ℃ of Refrigerator stores standby.
7. gained DNA liquid carries out electrophoresis detection with 1.5% sepharose, and detects its concentration with nucleic acid-protein instrument NanoVue Plus, and result is referring to Fig. 1 and table 1, the DNA electrophoresis result that Fig. 1 extracts for the method that the embodiment of the present invention provides, and wherein, M is Marker; The nucleic acid-protein instrument detected result of the DNA that table 1 provides for the embodiment of the present invention and comparative example.As shown in Figure 1, the band of the DNA of method extraction duck blood provided by the invention is bright single.
Comparative example 1
In precious biotechnology (Dalian) company limited, buy test kit TaKaRa MiniBEST Whole Blood Genomic DNA Extraction Kit.According to test kit operation instructions, extract fresh anti-freezing goose blood sample DNA, and carry out electrophoresis detection with 1.5% sepharose, and detect its concentration with nucleic acid-protein instrument NanoVue Plus, result is referring to Fig. 2 and table 1, wherein, and the DNA electrophoresis result that Fig. 2 extracts for the method that the embodiment of the present invention and comparative example provide, wherein, M is Marker, and E is embodiment 1, and e is comparative example 1; The nucleic acid-protein instrument detected result of the DNA that table 1 provides for the embodiment of the present invention and comparative example.As shown in Figure 2, the band of the DNA of method extraction goose blood provided by the invention is bright single, the DNA that its brightness is extracted higher than test kit.
Comparative example 2
In precious biotechnology (Dalian) company limited, buy test kit TaKaRa MiniBEST Whole Blood Genomic DNA Extraction Kit.According to test kit operation instructions, extract fresh anti-freezing chicken blood sample DNA, and carry out electrophoresis detection with 1.5% sepharose, and detect its concentration with nucleic acid-protein instrument NanoVue Plus, result is referring to Fig. 2 and table 1, wherein, and the DNA electrophoresis result that Fig. 2 extracts for the method that the embodiment of the present invention and comparative example provide, wherein, M is Marker, and J is embodiment 2, and j is comparative example 2; The nucleic acid-protein instrument detected result of the DNA that table 1 provides for the embodiment of the present invention and comparative example.As shown in Figure 2, the band of the DNA of method extraction chicken blood provided by the invention is bright single, the DNA that its brightness is extracted higher than test kit.
Comparative example 3
In precious biotechnology (Dalian) company limited, buy test kit TaKaRa MiniBEST Whole Blood Genomic DNA Extraction Kit.According to test kit operation instructions, extract fresh anti-freezing pigeon blood sample DNA, and carry out electrophoresis detection with 1.5% sepharose, and detect its concentration with nucleic acid-protein instrument NanoVue Plus, result is referring to Fig. 2 and table 1, wherein, and the DNA electrophoresis result that Fig. 2 extracts for the method that the embodiment of the present invention and comparative example provide, wherein, M is Marker, and G is embodiment 3, and g is comparative example 3; The nucleic acid-protein instrument detected result of the DNA that table 1 provides for the embodiment of the present invention and comparative example.As shown in Figure 2, the band of the DNA of method extraction pigeon blood provided by the invention is bright single, the DNA that its brightness is extracted higher than test kit.
Comparative example 4
In precious biotechnology (Dalian) company limited, buy test kit TaKaRa MiniBEST Whole Blood Genomic DNA Extraction Kit.According to test kit operation instructions, extract fresh anti-freezing duck blood sample DNA, and carry out electrophoresis detection with 1.5% sepharose, and detect its concentration with nucleic acid-protein instrument NanoVue Plus, result is referring to Fig. 2 and table 1, wherein, and the DNA electrophoresis result that Fig. 2 extracts for the method that the embodiment of the present invention and comparative example provide, wherein, M is Marker, and Y is embodiment 4, and y is comparative example 4; The nucleic acid-protein instrument detected result of the DNA that table 1 provides for the embodiment of the present invention and comparative example.As shown in Figure 2, the band of the DNA of method extraction duck blood provided by the invention is bright single, the DNA that its brightness is extracted higher than test kit.
The nucleic acid-protein instrument detected result of the DNA that table 1 embodiment of the present invention and comparative example provide
As shown in Table 1, method provided by the invention is suitable with kit method gained DNA quality, and even purity and receipts amount are higher than kit method.
Embodiment 5
The goose genomic dna that the embodiment 1 of take obtains is template, amplification goose endogenous reverse transcription II viroid (Genbank accession number:AY820076.1).
Pcr amplification system is 25uL:DNA template 2uL, 2 * Taq MasterMix (being purchased from Tian Gen biochemical technology company limited) 12.5uL, and each 0.75ul of upstream and downstream primer, complementary amount distilled water is to 25uL.
PCR reaction amplification program is: 95 ℃ of 5min of denaturation, circulate 34 times (94 ℃ of 30s of sex change, the 62 ℃ of 30s that anneal, extend 72 ℃ of 1min), and extend 72 ℃ of 5min, 4 ℃ of preservations.
Primer is:
F：5’-GCCCTAAAGGACTGTCCCAG-3’，
R：5’-CGCAAAGCTTCTGCAACGTA-3’。
62 ℃ of annealing temperatures.
Result is referring to Fig. 3, and Fig. 3 is the DNA profiling pcr amplification result that the embodiment of the present invention 1 is obtained, and wherein, M is Marker, and p is goose endogenous reverse transcription II viroid part amplified fragments.As shown in Figure 3, the embodiment 1 of take extracts the DNA obtaining and carries out pcr amplification as template, and PCR result band is bright, without assorted band, pollution-free, shows that the inventive method is extracted DNA quality good, can be used for Molecular Detection.
Embodiment 6
According to the method identical with embodiment 1, carry out DNA extraction, difference is not add Proteinase K solution, and result is referring to Fig. 4, the DNA electrophoresis result that Fig. 4 extracts for the method that the embodiment of the present invention 6～10 provides, wherein, M is Marker, E1 is embodiment 1, and E0 is embodiment 6.As shown in Figure 4, the band that does not add Proteinase K solution digestion adds the slightly dark of Proteinase K, but brightness is higher than maker, still can be for regular-PCR.
Embodiment 7
According to the method identical with embodiment 2, carry out DNA extraction, difference is not add Proteinase K solution, and result is referring to Fig. 4, the DNA electrophoresis result that Fig. 4 extracts for the method that the embodiment of the present invention 6～10 provides, wherein, M is Marker, J1 is embodiment 2, and J0 is embodiment 7.As shown in Figure 4, the band that does not add Proteinase K solution digestion adds the slightly dark of Proteinase K, but brightness is higher than maker, still can be for regular-PCR.
Embodiment 8
According to the method identical with embodiment 3, carry out DNA extraction, difference is not add Proteinase K solution, and result is referring to Fig. 4, the DNA electrophoresis result that Fig. 4 extracts for the method that the embodiment of the present invention 6～10 provides, wherein, M is Marker, G1 is embodiment 3, and G0 is embodiment 8.As shown in Figure 4, the band that does not add Proteinase K solution digestion adds the slightly dark of Proteinase K, but brightness is higher than maker, still can be for regular-PCR.
Embodiment 9
According to the method identical with embodiment 4, carry out DNA extraction, difference is not add Proteinase K solution, and result is referring to Fig. 4, the DNA electrophoresis result that Fig. 4 extracts for the method that the embodiment of the present invention 6～10 provides, wherein, M is Marker, Y1 is embodiment 4, and Y0 is embodiment 9.As shown in Figure 4, the band that does not add Proteinase K solution digestion adds the slightly dark of Proteinase K, but brightness is higher than maker, still can be for regular-PCR.
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 (10)
1. for extracting a cell pyrolysis liquid of poultry DNA, it is characterized in that, comprising:
0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 20mL/L～30mL/L Triton X-100; The water of surplus; With NaOH, regulating pH value is 8.
2. cell pyrolysis liquid according to claim 1, is characterized in that, comprising:
1.576g/L Tris-HCl; 0.7455g/L KCl; 2.033g/L MgCl
2; 0.7448g/L EDTA; 25mL/L Triton X-100; The water of surplus; With NaOH, regulating pH value is 8.
3. for extracting a cell damping fluid of poultry DNA, it is characterized in that, comprising:
0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 15g/L～30g/L NaCl; 0.5g/L～1.5g/L SDS; The water of surplus; With NaOH, regulating pH value is 8.
4. cell damping fluid according to claim 3, is characterized in that, comprising:
1.576g/L Tris-HCl; 0.7455g/L KCl; 2.033g/L MgCl
2; 0.7448g/L EDTA; 23.376g/L NaCl; 1g/L SDS; The water of surplus; With NaOH, regulating pH value is 8.
5. for extracting a cell pyrolysis liquid of poultry DNA, it is characterized in that, comprise Buffer WY and Buffer YW, wherein,
Described Buffer WY comprises: 0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 20mL/L～30mL/L Triton X-100; The water of surplus; With NaOH, regulating pH value is 8;
Described Buffer YW comprises: 0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 15g/L～30g/L NaCl; 0.5g/L～1.5g/L SDS; The water of surplus; With NaOH, regulating pH value is 8.
6. cell pyrolysis liquid according to claim 5, is characterized in that, described Buffer WY comprises: 1.576g/L Tris-HCl; 0.7455g/L KCl; 2.033g/L MgCl
2; 0.7448g/L EDTA; 25mL/L Triton X-100; The water of surplus; With NaOH, regulating pH value is 8;
Described Buffer YW comprises: 1.576g/L Tris-HCl; 0.7455g/L KCl; 2.033g/L MgCl
2; 0.7448g/L EDTA; 23.376g/L NaCl; 1g/L SDS; The water of surplus; With NaOH, regulating pH value is 8.
7. the poultry DNA extraction test kit that contains the cell pyrolysis liquid described in claim 5 or 6.
8. test kit according to claim 7, is characterized in that, also comprises: NaCl solution, Virahol, 75% alcohol and TE lysate.
9. a poultry DNA extraction method, comprising:
A) fresh anti-freezing poultry blood sample is mixed with Buffer WY solution, centrifugal after heating in water bath, by abandoning the material obtaining after supernatant, mix with Buffer WY solution, abandon supernatant after centrifugal to be precipitated; Described Buffer WY solution comprises: 0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 20mL/L～30mL/L Triton X-100; The water of surplus; With NaOH, regulating pH value is 8;
B) precipitation described step a) being obtained is mixed with Buffer YW solution and Proteinase K solution, adds NaCl solution after heating in water bath, centrifugal after purifying in nucleic acid purification post, obtains filtrate; Described Buffer YW solution comprises: 0.5g/L～2.5g/L Tris-HCl; 0.3g/L～1.0g/L KCl; 1.5g/L～2.5g/L MgCl
2; 0.5g/L～1.5g/L EDTA; 15g/L～30g/L NaCl; 0.5g/L～1.5g/L SDS; The water of surplus; With NaOH, regulating pH value is 8;
C) by described step b) filtrate that obtains mixes with Virahol, abandons supernatant after centrifugal, then mixes with 75% alcohol, abandons supernatant after centrifugal, adds TE lysate to dissolve after heating.
10. extracting method according to claim 10, it is characterized in that, described step a) is specially: the fresh anti-freezing poultry of 10～20 μ L blood sample is mixed with 1000 μ L Buffer WY solution concussions, centrifugal 1～the 3min of 10000～15000rpm/min after 60～70 ℃ of heating in water bath 5min～8min, by abandoning the material obtaining after supernatant, mix with 1000 μ L BufferWY solution concussions, after the centrifugal 1～3min of 10000～15000rpm/min, abandon supernatant and be precipitated;
Described step b) be specially: the precipitation that described step a) is obtained is mixed with the Proteinase K solution concussion of 200 μ L Buffer YW solution and 10 μ L20mg/mL, after 60～70 ℃ of heating in water bath 20min～30min, add the NaCl solution of 100 μ L5mol/L, in nucleic acid purification post, the centrifugal 1min of 6000～10000rpm/min after purifying, obtains filtrate;
Described step c) being specially: by described step b) filtrate that obtains mixes with isopyknic Virahol, after the centrifugal 3～5min of 10000～15000rpm/min, abandon supernatant, then mix with 1000～1500 μ L75% alcohol vibrations, after the centrifugal 1～3min of 10000～15000rpm/min, abandon supernatant, after 70～75 ℃ of baking oven for heating 1～3min, add 50mLTE lysate to dissolve.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108124451A (en) * | 2015-07-24 | 2018-06-05 | 塞弗德公司 | For extracting the composition and method of DNA and RNA from tissue sample |
| CN109852607A (en) * | 2018-12-30 | 2019-06-07 | 上海星耀医学科技发展有限公司 | It the reagent of erythroplastid and is applied in DNA extraction in a kind of removal biological sample |
| CN112626174A (en) * | 2020-12-16 | 2021-04-09 | 广州源井生物科技有限公司 | Cell lysis solution and application thereof |
| CN114790454A (en) * | 2022-03-02 | 2022-07-26 | 岭南现代农业科学与技术广东省实验室云浮分中心 | Kit and method for high-throughput full-automatic chicken blood DNA efficient extraction |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215558A (en) * | 2008-01-15 | 2008-07-09 | 陈刚 | Method for extracting whole blood DNA |
| CN101748119A (en) * | 2010-01-07 | 2010-06-23 | 四川农业大学 | Method for extracting whole blood DNA of bird species |
| CN102660537A (en) * | 2012-04-27 | 2012-09-12 | 云南农业大学 | Method for extracting a large number of high-purity deoxyribonucleic acid (DNA) from chicken blood |
| CN102690805A (en) * | 2011-12-01 | 2012-09-26 | 河南科技大学 | Method for rapidly extracting poultry blood genome deoxyribonucleic acid (DNA) |
| CN103882006A (en) * | 2012-12-19 | 2014-06-25 | 河北省健海生物芯片技术有限责任公司 | Preparation method and extraction method for DNA extraction reagent suitable for various samples |
| CN103898096A (en) * | 2014-03-27 | 2014-07-02 | 江苏佰龄全基因生物医学技术有限公司 | Mammal blood genome DNA extraction kit and method for extracting mammal blood genome DNA |
-
2014
- 2014-08-15 CN CN201410403604.0A patent/CN104152438B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215558A (en) * | 2008-01-15 | 2008-07-09 | 陈刚 | Method for extracting whole blood DNA |
| CN101748119A (en) * | 2010-01-07 | 2010-06-23 | 四川农业大学 | Method for extracting whole blood DNA of bird species |
| CN102690805A (en) * | 2011-12-01 | 2012-09-26 | 河南科技大学 | Method for rapidly extracting poultry blood genome deoxyribonucleic acid (DNA) |
| CN102660537A (en) * | 2012-04-27 | 2012-09-12 | 云南农业大学 | Method for extracting a large number of high-purity deoxyribonucleic acid (DNA) from chicken blood |
| CN103882006A (en) * | 2012-12-19 | 2014-06-25 | 河北省健海生物芯片技术有限责任公司 | Preparation method and extraction method for DNA extraction reagent suitable for various samples |
| CN103898096A (en) * | 2014-03-27 | 2014-07-02 | 江苏佰龄全基因生物医学技术有限公司 | Mammal blood genome DNA extraction kit and method for extracting mammal blood genome DNA |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108124451A (en) * | 2015-07-24 | 2018-06-05 | 塞弗德公司 | For extracting the composition and method of DNA and RNA from tissue sample |
| CN109852607A (en) * | 2018-12-30 | 2019-06-07 | 上海星耀医学科技发展有限公司 | It the reagent of erythroplastid and is applied in DNA extraction in a kind of removal biological sample |
| CN112626174A (en) * | 2020-12-16 | 2021-04-09 | 广州源井生物科技有限公司 | Cell lysis solution and application thereof |
| CN114790454A (en) * | 2022-03-02 | 2022-07-26 | 岭南现代农业科学与技术广东省实验室云浮分中心 | Kit and method for high-throughput full-automatic chicken blood DNA efficient extraction |
| CN114790454B (en) * | 2022-03-02 | 2024-09-06 | 温氏食品集团股份有限公司 | Kit and method for high-throughput full-automatic chicken blood DNA efficient extraction |
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