CN101948828B - Trace animal cell genomic DNA template preparation solution and corresponding DNA template preparation method - Google Patents
Trace animal cell genomic DNA template preparation solution and corresponding DNA template preparation method Download PDFInfo
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- CN101948828B CN101948828B CN2010105070708A CN201010507070A CN101948828B CN 101948828 B CN101948828 B CN 101948828B CN 2010105070708 A CN2010105070708 A CN 2010105070708A CN 201010507070 A CN201010507070 A CN 201010507070A CN 101948828 B CN101948828 B CN 101948828B
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- genomic dna
- dna template
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- zooblast
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- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 210000004102 animal cell Anatomy 0.000 title abstract 4
- 239000000243 solution Substances 0.000 claims abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 239000007983 Tris buffer Substances 0.000 claims abstract description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims abstract description 6
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims description 44
- 238000009413 insulation Methods 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 11
- 108010067770 Endopeptidase K Proteins 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 2
- 230000001476 alcoholic effect Effects 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 6
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 abstract description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 abstract 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 abstract 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 abstract 2
- 235000011130 ammonium sulphate Nutrition 0.000 abstract 2
- 229910001629 magnesium chloride Inorganic materials 0.000 abstract 2
- 239000011259 mixed solution Substances 0.000 abstract 2
- 239000002699 waste material Substances 0.000 abstract 1
- 238000000034 method Methods 0.000 description 13
- 239000012153 distilled water Substances 0.000 description 9
- 238000011144 upstream manufacturing Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 8
- 210000001771 cumulus cell Anatomy 0.000 description 6
- 210000003746 feather Anatomy 0.000 description 6
- 210000004209 hair Anatomy 0.000 description 6
- 210000000287 oocyte Anatomy 0.000 description 6
- 238000012408 PCR amplification Methods 0.000 description 5
- 210000003443 bladder cell Anatomy 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 229920002684 Sepharose Polymers 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 238000004043 dyeing Methods 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 235000019833 protease Nutrition 0.000 description 3
- 241000283725 Bos Species 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 210000003780 hair follicle Anatomy 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 210000000442 hair follicle cell Anatomy 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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Abstract
The invention belongs to the field of molecular biology and discloses trace animal cell genomic DNA template preparation solution and a corresponding DNA template preparation method. The trace animal cell genomic DNA template preparation solution is prepared by mixing solution A and solution B in a volume ratio of 1:3-10, wherein the solution A is the mixed solution of Tris, (NH4)2SO4 and MgCl2, and the final concentration of the Tris, the (NH4)2SO4 and the MgCl2 is 0.1 to 2.0M respectively; and the solution B is the mixed solution of Triton and beta-mercaptoethanol, and the final concentration of the Triton and the beta-mercaptoethanol is 0.1 to 2.0 percent respectively. Compared with the cost of the conventional reagent required for extracting genomic DNA, the cost of the trace animal cell genomic DNA template preparation solution is greatly reduced; and as the template preparation process is simple, the prepared genomic DNA template is complete, waste is avoided, and the cell genomic DNA template can be successfully prepared at one time.
Description
Technical field
The invention belongs to biology field, relate to a kind of micro-zooblast genomic dna template and prepare liquid and corresponding D NA method for preparing template.
Background technology
Existing zooblast genome DNA extracting method is a lot, comprise phenol chloroform extraction method etc., leaching process is loaded down with trivial details owing to processes such as the DNA purifying need wash, and because the yield problem of DNA purifying, need the initial sample size of cell more, therefore generally need to extract the animal blood sample or adopt animal tissues's sample, animal is damaged bigger.Existing micro-zooblast genome DNA extracting reagent kit is owing to needing the purifying pillar to cost an arm and a leg on the market, and process is also loaded down with trivial details relatively.
Summary of the invention
The object of the invention is the deficiency that exists at existing zooblast extracting genome DNA is provided, and provides a kind of micro-zooblast genomic dna template to prepare liquid and corresponding D NA method for preparing template.
A kind of micro-zooblast genomic dna template prepares liquid, and this preparation liquid is got so that the volume ratio of 1:3-10 is mixed by A liquid and B liquid, and wherein said A liquid is Tris, (NH
4)
2SO
4, MgCl
2Mixing solutions, three kinds of reagent final concentrations are 0.1-2.0M; Described B liquid is Triton and β-coloured glaze base alcoholic acid mixing solutions, and two kinds of reagent final concentrations are 0.1-2.0%.。
Corresponding D NA method for preparing template in turn includes the following steps:
(1) gets above-mentioned micro-zooblast genomic dna template and prepare liquid, add and be equipped with in the centrifuge tube or PCR pipe of micro-zooblast;
(2) described dna profiling will be housed and prepare the centrifuge tube of liquid and zooblast or 95 ℃ of insulations of PCR pipe 5 minutes, 16 ℃ of insulations 5 minutes;
(3) add Proteinase K solution, this Proteinase K final concentration is 1.0-3.0mg/ml;
(4) 55 ℃ the insulation 2 hours or more than, 95 ℃ the insulation 10 minutes, 16 ℃ the insulation 5 minutes;
(5) centrifuging and taking supernatant, this supernatant liquor directly are used as the dna profiling of PCR.
Described dna profiling preparation method preferably in turn includes the following steps:
(1) gets above-mentioned micro-zooblast genomic dna template and prepare liquid 8.8 μ l, add and be equipped with in the centrifuge tube or PCR pipe of zooblast;
(2) described dna profiling will be housed and prepare the centrifuge tube of liquid and zooblast or 95 ℃ of insulations of PCR pipe 5 minutes, 16 ℃ of insulations 5 minutes;
(3) adding concentration is 10mg/ml Proteinase K solution 1.2 μ l, and making this Proteinase K final concentration is 1.2mg/ml, obtains total reaction volume 10 μ l;
(4) 55 ℃ the insulation 2 hours or more than, 95 ℃ the insulation 10 minutes, 16 ℃ the insulation 5 minutes;
(5) centrifuging and taking supernatant, this supernatant liquor directly are used as the dna profiling of PCR.
Beneficial effect:
The present invention's trace zooblast genomic dna template prepares the liquid cost and significantly reduces than the required reagent cost of existing zooblast extracting genome DNA.Zooblast genomic dna template preparation process of the present invention is simple, utilize the dna profiling of being prepared to prepare liquid directly with the zooblast cracking, the DNA that discharges need not to carry out purifying and can avoid ordinary method to extract the complicated processes of genomic dna directly as the dna profiling of conventional PCR.Because the template preparation process is simple, the present invention is complete from the genomic dna template of micro-zooblast preparation, not consume.The template of the present invention's trace zooblast genomic dna prepares liquid and corresponding D NA method for preparing template is less because of required initial zooblast quantity, can be used for the preparation of the genomic dna templates such as animal culturing cell of animal hair hair follicle, denier, make extensive zooblast genomic dna template preparation become possibility.Because it is sampling such as animal hair is simple, than the animal blood sample or organize the sampling means of sample etc., littler to the injury of animal.
Description of drawings
Fig. 1, embodiment 3 utilize the present invention to prepare squab trace feather pulp cell genomic dna template and squab male and female identification result figure,
M：Marker?DL?2000。
Fig. 2, embodiment 4 utilize the present invention to prepare the PCR qualification result figure of ox hair bladder cell genomic dna template,
M：Marker?DL?2000。
Fig. 3, embodiment 5 utilize the present invention to prepare the single ovocyte periphery of the pig PCR qualification result figure of cumulus cell genomic dna template on a small quantity,
M:Marker DL 2000, swimming lane 1,2: genomic dna amplified fragments, swimming lane 3,4: negative control.
Embodiment
Embodiment 1The preparation that trace zooblast genomic dna template prepares liquid
A liquid: Tris, (NH
4)
2SO
4, MgCl
2Three kinds of reagent are dissolved in distilled water, make its final concentration be 0.1M;
B liquid: Triton and beta-mercaptoethanol dilute with distilled water, make its final concentration be 0.1%;
With the distilled water is solvent, prepares A liquid and B liquid according to a conventional method respectively, and the ratio with 1:3 is even with two kinds of liquid mixing of A, B then, promptly gets zooblast genomic dna template and prepares liquid.
Embodiment 2The preparation that trace zooblast genomic dna template prepares liquid
A liquid: Tris, (NH
4)
2SO
4, MgCl
2Three kinds of reagent are dissolved in distilled water, make its final concentration be 2.0M;
B liquid: Triton and beta-mercaptoethanol dilute with distilled water, make its final concentration be 2.0%;
With the distilled water is solvent, prepares A liquid and B liquid according to a conventional method respectively, and the ratio with 1:10 is even with two kinds of liquid mixing of A, B then, promptly gets zooblast genomic dna template and prepares liquid.
Embodiment 3Utilize squab trace feather pulp cell preparation genomic dna template and squab male and female to differentiate
1.1 squab feather pulp cell genomic dna template preparation
The newborn veutro feather of 7 days left and right sides of allocation squabs (containing a small amount of feather pulp) 1, and get 1mg left and right sides feather pulp in the PCR pipe;
Add embodiment 1 micro-zooblast genomic dna template in the PCR pipe and prepare liquid 8.8 μ l;
The PCR pipe put in the PCR instrument 95 ℃ 5 minutes, 16 ℃ 5 minutes, 55 ℃ 2 hours, 95 ℃ 10 minutes, 16 ℃ 5 minutes.And in first 16 ℃ of 5 minutes mistakes are planted, suspend the PCR instrument, add 1.2 μ l Proteinase Ks (10mg/ml);
The centrifuging and taking supernatant is made the dna profiling of PCR.
1.2 pcr amplification
Reaction system: distilled water 6.05 μ l, 25mM Mg
2+1.0 μ l, 10 * Buffer, 1.0 μ l, 5mM dNTP 0.4 μ l, 5 μ M upstream and downstream primers each 0.5 μ l, Taq enzyme 0.05 μ l, dna profiling 0.5 μ l; Upstream primer is: SEQ ID NO.1, downstream primer is: SEQ ID NO.2.
Reaction conditions: 94 ℃ 2 minutes; 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 40 seconds, 35 circulations.
1.3 PCR product band somatotype
EB dyeing, 1% sepharose 5-10V/cm electrophoresis, imaging system band somatotype.Female dove is three bands, and length is respectively 628bp, 420bp and 358bp; Male dove is two bands, and length is respectively 628bp and 358bp(sees Fig. 1).
Embodiment 4Utilize the ox hair bladder cell to prepare the genomic dna template
1.1 breeding oxen hair follicle cell genomic dna template preparation
1 in allocation breeding oxen hair, and get hair follicle in the PCR pipe;
Add embodiment 2 micro-zooblast genomic dna templates in the PCR pipe and prepare liquid 8.8 μ l;
The PCR pipe put in the PCR instrument 95 ℃ 5 minutes, 16 ℃ 5 minutes, 55 ℃ 2 hours, 95 ℃ 10 minutes, 16 ℃ 5 minutes.And in first 16 ℃ of 5 minutes mistakes are planted, suspend the PCR instrument, add 1.2 μ l Proteinase Ks (10 mg/ml);
The centrifuging and taking supernatant is made the dna profiling of PCR.
1.2 pcr amplification
Reaction system: distilled water 6.05 μ l, 25mM Mg
2+1.0 μ l, 10 * Buffer, 1.0 μ l, 5mM dNTP 0.4 μ l, the described upstream and downstream of 5 μ M primer each 0.5 μ l, Taq enzyme 0.05 μ l, dna profiling 0.5 μ l; Upstream primer is: SEQ ID NO.3, downstream primer is: SEQ ID NO.4.
Reaction conditions: 94 ℃ 2 minutes; 94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 40 seconds, 35 circulations.
1.3 the PCR of genomic dna template identifies
EB dyeing, 1% sepharose 5-10V/cm electrophoresis, imaging system is identified pcr amplification product band (see figure 2).
Embodiment 5Utilize a small amount of cumulus cell of the single ovocyte periphery of pig to prepare the genomic dna template
1.1 a small amount of cumulus cell genomic dna template preparation of single ovocyte periphery
Draw single ovocyte (containing a small amount of cumulus cell), and be blown in the PCR pipe (PBS is few as far as possible);
Add embodiment 2 micro-zooblast genomic dna templates in the PCR pipe and prepare liquid 8.8 μ l;
The PCR pipe put in the PCR instrument 95 ℃ 5 minutes, 16 ℃ 5 minutes, 55 ℃ 2 hours, 95 ℃ 10 minutes, 16 ℃ 5 minutes.And in first 16 ℃ of 5 minutes mistakes are planted, suspend the PCR instrument, add 1.2 μ l Proteinase Ks (10mg/ml);
The centrifuging and taking supernatant is made the dna profiling of PCR.
1.2 pcr amplification
Reaction system: distilled water 6.05 μ l, 25mM Mg
2+1.0 μ l, 10 * Buffer, 1.0 μ l, 5mM dNTP 0.4 μ l, the described upstream and downstream of 5 μ M primer each 0.5 μ l, Taq enzyme 0.05 μ l, dna profiling 0.5 μ l; Upstream primer is: SEQ ID NO.5, downstream primer is: SEQ ID NO.6.
Reaction conditions: 94 ℃ 2 minutes; 94 ℃ 30 seconds, 59 ℃ 30 seconds, 72 ℃ 30 seconds, 35 circulations.
1.3 the PCR of genomic dna template identifies
EB dyeing, 1% sepharose 5-10V/cm electrophoresis, imaging system is identified pcr amplification product band (see figure 3).
＜110〉Jiangsu Province Agriculture Science Institute
＜120〉a kind of micro-zooblast genomic dna template prepares liquid and corresponding D NA method for preparing template
<160>?6
<210>?1
<211>?18
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉the squab male and female are differentiated PCR upstream primer sequence
<400>?1
aacgtggcaa?cagagtac 18
<210>?2
<211>?18
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉the squab male and female are differentiated PCR downstream primer sequence
<400>2
gatccagtgc?ttgtttcc 18
<210>?3
<211>?19
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉PCR of ox hair bladder cell genomic dna template identifies the upstream primer sequence
<400>3
agcatcacag?cagctggac 19
<210>?4
<211>?20
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉PCR of ox hair bladder cell genomic dna template identifies the downstream primer sequence
<400>4
gctttccatc?caagtacgag 20
<210>?5
<211>?22
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉PCR of a small amount of cumulus cell genomic dna template of the single ovocyte periphery of pig identifies the upstream primer sequence
<400>5
aagtcggaag?gtcattgtcg?tg 22
<210>?6
<211>?21
<212>?DNA
＜213〉artificial sequence
<220>
＜223〉PCR of a small amount of cumulus cell genomic dna template of the single ovocyte periphery of pig identifies the downstream primer sequence
<400>?6
cgcccagtcc?aggtaggtat?t 21
Claims (3)
1. a micro-zooblast genomic dna template prepares liquid, it is characterized in that this preparation liquid by A liquid and B liquid with 1: the volume ratio of 3-10 is mixed, and wherein said A liquid is Tris, (NH
4)
2SO
4, MgCl
2Mixing solutions, three kinds of reagent final concentrations are 0.1-2.0M; Described B liquid is Triton and β-coloured glaze base alcoholic acid mixing solutions, and two kinds of reagent final concentrations are 0.1-2.0%.
2. dna profiling preparation method is characterized in that in turn including the following steps:
(1) the weighting profit requires 1 described micro-zooblast genomic dna template to prepare liquid, adds to be equipped with in the centrifuge tube or PCR pipe of micro-zooblast;
(2) described dna profiling will be housed and prepare the centrifuge tube of liquid and zooblast or PCR pipe, 16 ℃ of insulations 5 minutes in 95 ℃ of insulations 5 minutes;
(3) add Proteinase K solution, making this Proteinase K final concentration is 1.0-3.0mg/ml;
(4) 55 ℃ the insulation 2 hours or more than, 95 ℃ the insulation 10 minutes, 16 ℃ the insulation 5 minutes;
(5) centrifuging and taking supernatant, this supernatant liquor directly are used as the dna profiling of PCR.
3. dna profiling preparation method according to claim 2 in turn includes the following steps:
(1) the weighting profit requires 1 described micro-zooblast genomic dna template to prepare liquid 8.8 μ l, adds to be equipped with in the centrifuge tube or PCR pipe of micro-zooblast;
(2) described dna profiling will be housed and prepare the centrifuge tube of liquid and zooblast or PCR pipe, 16 ℃ of insulations 5 minutes in 95 ℃ of insulations 5 minutes;
(3) adding concentration is 10mg/ml Proteinase K solution 1.2 μ l, and making this Proteinase K final concentration is 1.2mg/ml, obtains total reaction volume 10 μ l;
(4) 55 ℃ the insulation 2 hours or more than, 95 ℃ the insulation 10 minutes, 16 ℃ the insulation 5 minutes;
(5) centrifuging and taking supernatant, this supernatant liquor directly are used as the dna profiling of PCR.
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| CN112094840A (en) * | 2019-06-17 | 2020-12-18 | 南京尧顺禹生物科技有限公司 | Rapid preparation method of trace animal sample genome DNA template for genome segment amplification |
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| CN101532011A (en) * | 2008-03-10 | 2009-09-16 | 首都师范大学 | Kit for rapidly extracting and purifying DNA/RNA from same sample and method thereof |
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| CN101532011A (en) * | 2008-03-10 | 2009-09-16 | 首都师范大学 | Kit for rapidly extracting and purifying DNA/RNA from same sample and method thereof |
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| Title |
|---|
| 叶冰莹.阔叶榧总DNA的提取及其RAPD反应条件的研究.《厦门大学学报》.2002,第41卷(第5期),683-686. * |
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