CN103266107B - A kind of extracting method of arid biogeographic zone fruit tree crop genomic dna - Google Patents
A kind of extracting method of arid biogeographic zone fruit tree crop genomic dna Download PDFInfo
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Abstract
The present invention relates to a kind of extracting method of arid biogeographic zone fruit tree crop genomic dna, the method is from arid biogeographic zone fruit tree plant leaf tissue, particularly be rich in the fruit tree plant leaf tissue of polysaccharide polyphenol or starch and pass through liquid nitrogen grinding, proceed in centrifuge tube, add Extraction buffer, temperature bath in water-bath, centrifugal, Aspirate supernatant, supernatant liquor is proceeded in two other new centrifuge tube, add chloroform and primary isoamyl alcohol respectively, centrifugal, Aspirate supernatant again, merge supernatant liquor, proceed in another new centrifuge tube, add chloroform and primary isoamyl alcohol, centrifugal, Aspirate supernatant again, supernatant liquor is proceeded in another new centrifuge tube, add freezing dehydrated alcohol, put into refrigerator, centrifugal, there is clear milky DNA white cotton fiber precipitation, by washing with alcohol, after natural air drying, be dissolved in buffered soln, Refrigerator store can obtain high-quality fruit tree genomic dna, be can be used in the experiments such as follow-up pcr amplification and molecular cloning by method of the present invention.For the utilization of further Protocols in Molecular Biology on arid biogeographic zone fruit tree is laid a good foundation.
Description
Technical field
The present invention relates to a kind of extracting method being applicable to arid biogeographic zone fruit tree crop genomic dna.
Background technology
Xinjiang is located in Eurasian innerland, it is typical continental climate, sunshine duration long (year sunshine time 2500-3500 hour), little (the average rainfall 145mm of rainfall amount, North SinKiang 200mm, South Sinkiang is less than 100mm), day and night temperature large (average temperature of the whole year diurnal range about 15 DEG C), atmospheric moisture is little, form a unique weather group, just because of the weather of this uniqueness, the physical environment of that Xinjiang is possessed is advantageous development fruit tree, particularly Southern Xinjiang, belong to typical arid biogeographic zone, enrich the photosynthesis enhancing fruit tree sunshine, the temperature difference is beneficial to greatly the accumulation of carbohydrate, make fruit sugar degree high, but, incidently be, the fruit tree of arid biogeographic zone cultivation is (as apple, pears, apricot, peach, jujube, walnut, almond, Pistacia vera etc.) tree body and blade be rich in phenols, carbohydrate, the secondary metabolites such as pigment and tannin, this brings larger difficulty to the high-quality fruit tree genomic dna of acquisition, DNA marker is carried out in the extraction of genomic dna, build the basis of genomic library and the biological study of genetic map equimolecular.
The DNA quantity that the method using the CTAB method of normal conventional to extract DNA obtains is few, and purity is low, is difficult to carry out pcr amplification, and then affects the application of follow-up all kinds of Protocols in Molecular Biology on fruit tree material.For this problem, the present invention improves CTAB method, from arid biogeographic zone fruit tree plant leaf tissue, particularly be rich in rapid extraction genome DNA in the fruit tree plant leaf tissue of polysaccharide polyphenol or starch, obtain high-quality fruit tree genomic dna, establish a set of method that is stable, applicable arid biogeographic zone fruit tree material DNA rapid extraction efficiently, and can be used in the experiments such as follow-up pcr amplification and molecular cloning.For the utilization of further Protocols in Molecular Biology on arid biogeographic zone fruit tree is laid a good foundation.
Summary of the invention
The object of the invention is, a kind of extracting method being applicable to arid biogeographic zone fruit tree crop genomic dna is provided, the method is from arid biogeographic zone fruit tree plant leaf tissue, particularly be rich in the fruit tree plant leaf tissue of polysaccharide polyphenol or starch and pass through liquid nitrogen grinding, proceed in centrifuge tube, add Extraction buffer, temperature bath in water-bath, centrifugal, Aspirate supernatant, supernatant liquor is proceeded in two other new centrifuge tube, add chloroform and primary isoamyl alcohol respectively, centrifugal, Aspirate supernatant again, merge supernatant liquor, proceed in another new centrifuge tube, add chloroform and primary isoamyl alcohol, centrifugal, Aspirate supernatant again, supernatant liquor is proceeded in another new centrifuge tube, add freezing dehydrated alcohol, put into refrigerator, centrifugal, there is clear milky DNA white cotton fiber precipitation, by washing with alcohol, after natural air drying, be dissolved in buffered soln, Refrigerator store can obtain high-quality fruit tree genomic dna, the present invention establishes a set of stable, be applicable to the method for arid biogeographic zone fruit tree material DNA rapid extraction efficiently, and can be used in the experiments such as follow-up pcr amplification and molecular cloning.For the utilization of further Protocols in Molecular Biology on arid biogeographic zone fruit tree is laid a good foundation.
The extracting method of a kind of arid biogeographic zone fruit tree crop genomic dna of the present invention, follows these steps to carry out:
A, take the blade of fruit tree material, be placed in the mortar after precooling, sterilizing, add liquid nitrogen grinding to powder, proceed in 1500 μ L centrifuge tubes;
B, add 1000 μ L rapidly, temperature 65 DEG C of preheatings containing w/v2% cetyl trimethylammonium bromide, 1.4M sodium-chlor, 20mM tetraacethyl diamino-vinyl, 100mM Tutofusin tris, the damping fluid of pH8.0 and 8% mercaptoethanol, shakes up gently;
C, be placed in temperature 65 DEG C of water-bath temperature bath 60min, teetertotter gently every 10min therebetween and shake up 1 time, make the abundant cracking of solution;
D, employing desk centrifuge are centrifugal, temperature 21 DEG C, rotating speed 10000r/min, time 15min, and Aspirate supernatant, proceeds in two other 1500 new μ L centrifuge tubes by supernatant liquor evenly distribute;
E, add isopyknic chloroform by each centrifuge tube containing supernatant liquor: primary isoamyl alcohol=24:1, teetertotter gently and shake up, centrifugal, temperature 21 DEG C, rotating speed 10000r/min, time 15min, Aspirate supernatant, supernatant liquor in each centrifuge tube is merged, proceeds in another 1500 new μ L centrifuge tubes;
F, add isopyknic chloroform by the centrifuge tube that 600 μ L supernatant liquors are housed: primary isoamyl alcohol=24:1, teetertotters gently and shake up, centrifugal, temperature 21 DEG C, rotating speed 10000r/min, time 15min, Aspirate supernatant again, proceeds in another 1500 new μ L centrifuge tubes by supernatant liquor;
G, by adding the freezing dehydrated alcohol of 2 times of volumes in the centrifuge tube that 400 μ L supernatant liquors are housed, put into temperature-20 DEG C of deep freezers, time 30min, centrifugal, temperature 21 DEG C, rotating speed 10000r/min, there is clear milky DNA white cotton fiber precipitation after time 15min, remove upper liquid;
H, again by milky DNA white cotton fiber precipitation concentration be 70% washing with alcohol 2-3 time, after natural air drying, be dissolved in 50 μ L and contain 10mM Tutofusin tris, 1mM tetraacethyl diamino-vinyl, in the buffered soln of pH8.0, temperature-20 DEG C of Refrigerator stores.
In steps d in each centrifuge tube all containing 480 μ L supernatant liquors.
600 μ L supernatant liquors are contained in centrifuge tube in step e.
400 μ L supernatant liquors are contained in centrifuge tube in step f.
Described method is applicable to the fruit tree of arid biogeographic zone cultivation as apple, pears, apricot, peach, jujube, walnut, almond, Pistacia vera.
Described method is extracted in the process of DNA arid biogeographic zone fruit tree plant, adopts material that is fresh, freezing or silica dehydrator preservation.
The arid biogeographic zone fruit tree crop genomic dna obtained by the method for the invention carries out quality examination:
Get 3 μ lDNA solution distilled waters and be diluted to 3000 μ l, measure its OD value at 260nm and 280nm with ultraviolet spectrophotometer (Eppendorf, Germany), purity estimation standard: pure DNA solution, its OD
260/ OD
280value=1.7 ~ 1.9; By the public output calculating DNA: output (ng/ μ L)=D260nm × 50 × extension rate, calculate the output of DNA;
Judge the quality and quantity of DNA in conjunction with 1% agarose gel electrophoresis, the quality and quantity of the upper electrophoresis detection DNA of the sepharose (EB dyeing) 0.8% simultaneously;
The Accurate Measurement of DNA output:
Take out the DNA of 50 μ l, be diluted to 100ng/ μ l;
Prepare the λ DNA standardized solution (200ng/ μ l, 150ng/ μ l, 100ng/ μ l and 50ng/ μ l) of following concentration respectively;
Label taking accurate λ DNA and each 1 μ l of arid biogeographic zone fruit tree crop genome DNA sample, carries out the agarose gel electrophoresis of 1%, and EB dyes observation;
The concentration of observation and comparison arid biogeographic zone fruit tree crop genome DNA sample and λ DNA standard model in BIO-RAD type UVP gel imaging system, thus carry out quantitatively to template DNA sample, with the brightness of 100ng λ DNA standard model for standard, through repeatedly adjusting the concentration of sample, until the brightness of its loading 1 μ l is consistent with the brightness of λ DNA standard model 100ng/ μ l.
Accompanying drawing explanation
Fig. 1 is arid biogeographic zone fruit tree leaf material genomic dna 1% agarose gel electrophoresis figure of the present invention, is wherein: apple, pears, apricot, peach, jujube, walnut, almond that the fruit trees such as Pistacia vera account for two swimming lanes respectively from left to right.
Embodiment
Embodiment 1
Take the blade 0.1g of fruit tree material, be placed in the mortar after precooling, sterilizing, add liquid nitrogen grinding to powder, proceed in 1500 μ L centrifuge tubes;
Add rapidly 1000 μ L, temperature 65 DEG C of preheatings containing w/v2% cetyl trimethylammonium bromide, 1.4M sodium-chlor, 20mM tetraacethyl diamino-vinyl, 100mM Tutofusin tris, the damping fluid of pH8.0 and 8% mercaptoethanol, shakes up gently;
Be placed in temperature 65 DEG C of water-bath temperature bath 60min, teetertotter gently every 10min therebetween and shake up 1 time, make the abundant cracking of solution;
Adopt the centrifugal (model: eppendorf-Centrifuge5804R of desk centrifuge, the place of production: Germany), temperature 21 DEG C, rotating speed 10000r/min, time 15min, Aspirate supernatant, proceeds to supernatant liquor evenly distribute in two other 1500 new μ L centrifuge tubes, all has 480 μ L supernatant liquors in each centrifuge tube;
Isopyknic chloroform is added containing in the centrifuge tube of supernatant liquor: primary isoamyl alcohol=24:1 by each, teetertotter gently and shake up, centrifugal, temperature 21 DEG C, rotating speed 10000r/min, time 15min, Aspirate supernatant, supernatant liquor in each centrifuge tube is merged, proceeds in another 1500 new μ L centrifuge tubes, wherein in centrifuge tube, about have 600 μ L supernatant liquors;
Isopyknic chloroform is added: primary isoamyl alcohol=24:1 by the centrifuge tube that 600 μ L supernatant liquors are housed, teetertotter gently and shake up, centrifugal, temperature 21 DEG C, rotating speed 10000r/min, time 15min, then Aspirate supernatant, supernatant liquor is proceeded in another 1500 new μ L centrifuge tubes, wherein in centrifuge tube, about have 400 μ L supernatant liquors;
By adding the freezing dehydrated alcohol of 2 times of volumes in the centrifuge tube that 400 μ L supernatant liquors are housed, put into temperature-20 DEG C of deep freezers, time 30min, centrifugal, temperature 21 DEG C, rotating speed 10000r/min, there is clear milky DNA white cotton fiber precipitation after time 15min, remove upper liquid;
Be washing with alcohol 2-3 time of 70% again by milky DNA white cotton fiber precipitation concentration, after natural air drying, be dissolved in 50 μ L and contain 10mM Tutofusin tris, 1mM tetraacethyl diamino-vinyl, in the buffered soln of pH8.0, temperature-20 DEG C of Refrigerator stores.Extract at arid biogeographic zone fruit tree plant in the process of DNA, adopt material that is fresh, freezing or silica dehydrator preservation.
Embodiment 2
The arid biogeographic zone fruit tree plant obtained by the method for the invention extracts the quality examination of DNA:
Get 3 μ L arid biogeographic zone fruit tree plants extraction DNA solution distilled waters and be diluted to 3000 μ L, with ultraviolet spectrophotometer (Eppendorf, Germany) its OD value (optical density(OD)) at 260nm and 280nm is measured, purity estimation standard: pure DNA solution, its OD
260/ OD
280value=1.7-1.9; By the public output calculating DNA: output (ng/ μ L)=D260nm × 50 × extension rate, calculate the output of DNA;
Judge that arid biogeographic zone fruit tree plant extracts the quality and quantity of DNA in conjunction with 1% agarose gel electrophoresis, the upper electrophoresis detection arid biogeographic zone fruit tree plant of the sepharose (EB dyeing) 0.8% extracts the quality and quantity of DNA simultaneously;
The Accurate Measurement of DNA output:
The arid biogeographic zone fruit tree plant taking out 50 μ L extracts DNA, is diluted to 100ng/ μ L;
Prepare the λ DNA standardized solution of a small amount of 200ng/ μ L, 150ng/ μ L, 100ng/ μ L and 50ng/ μ L;
Label taking accurate λ DNA and arid biogeographic zone fruit tree plant extract each 1 μ L of DNA sample, carry out the agarose gel electrophoresis of 1%, EB(ethidium bromide) dyeing observation;
In gel imaging system (model: BIO-RAD-UVP, the place of production: Germany) in the concentration of observation and comparison sample DNA and λ DNA standard model, thus carry out quantitatively to template DNA sample, with the brightness of 100ng λ DNA standard model for standard, through repeatedly adjusting the concentration of sample, until the brightness of its loading 1 μ L is consistent with the brightness of λ DNA standard model 100ng/ μ L;
The extraction of arid biogeographic zone fruit tree material genomic dna and quality evaluation table 1:
The detection of table 1DNA purity and output
As can be seen from the table: adopt 8 fruit tree species material genome DNA sample (apples that the CTAB method improved is extracted, pears, apricot, peach, jujube, walnut, almond, Pistacia vera), the genomic dna yield of all seeds is all higher, purity check shows that the D260nm/D280nm of the genomic dna of all samples is all between 1.70-1.95, shows that mentioned genomic dna purity is high; Their ultraviolet absorption curve is typical nucleic acid Absorption Characteristics, and occur at D258 ~ 260nm place absorbing peak, in appearance in white or colourless precipitate, jelly is few.
Detected through gel electrophoresis (Fig. 1) shows, and protein remaining in loading wells is little, and the DNA band carried is complete clear, less degradation, without serious traction phenomenon, DNA content purity is high, inclusion-free, DNA fragmentation is complete, and extraction effect is desirable, can be satisfied with the requirement of further molecular biology experiment.
Claims (1)
1. an extracting method for arid biogeographic zone fruit tree crop genomic dna, is characterized in that following these steps to carry out:
A, take fruit tree material as apple, pears, apricot, peach, jujube, walnut, almond, the blade of Pistacia vera, be placed in the mortar after precooling, sterilizing, add liquid nitrogen grinding to powder, proceed in 1500 μ L centrifuge tubes;
B, add 1000 μ L rapidly, temperature 65 DEG C of preheatings containing 2% cetyl trimethylammonium bromide, 1.4M sodium-chlor, 20mM tetraacethyl diamino-vinyl, 100mM Tutofusin tris, the damping fluid of pH8.0 and the damping fluid of 8% mercaptoethanol, shake up gently;
C, be placed in temperature 65 DEG C of water-bath temperature bath 60min, teetertotter gently every 10min therebetween and shake up 1 time, make the abundant cracking of solution;
D, adopt desk centrifuge centrifugal, temperature 21 DEG C, rotating speed 10000r/min, time 15min, Aspirate supernatant, proceeds in two other 1500 new μ L centrifuge tubes by supernatant liquor evenly distribute, wherein in each centrifuge tube all containing 480 μ L supernatant liquors;
E, add isopyknic chloroform by each centrifuge tube containing supernatant liquor: primary isoamyl alcohol=24:1, teetertotter gently and shake up, centrifugal, temperature 21 DEG C, rotating speed 10000r/min, time 15min, Aspirate supernatant, supernatant liquor in each centrifuge tube is merged, proceeds in another 1500 new μ L centrifuge tubes, wherein contain 600 μ L supernatant liquors in centrifuge tube;
F, add isopyknic chloroform by the centrifuge tube that 600 μ L supernatant liquors are housed: primary isoamyl alcohol=24:1, teetertotter gently and shake up, centrifugal, temperature 21 DEG C, rotating speed 10000r/min, time 15min, then Aspirate supernatant, supernatant liquor is proceeded in another 1500 new μ L centrifuge tubes, wherein contain 400 μ L supernatant liquors in centrifuge tube;
G, by adding the freezing dehydrated alcohol of 2 times of volumes in the centrifuge tube that 400 μ L supernatant liquors are housed, put into temperature-20 DEG C of deep freezers, time 30min, centrifugal, temperature 21 DEG C, rotating speed 10000r/min, there is clear milky DNA white cotton fiber precipitation after time 15min, remove upper liquid;
H, again by milky DNA white cotton fiber precipitation concentration be 70% washing with alcohol 2-3 time, after natural air drying, be dissolved in 50 μ L and contain 10mM Tutofusin tris, 1mM tetraacethyl diamino-vinyl, in the buffered soln of pH8.0, temperature-20 DEG C of Refrigerator stores.
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| CN102676496A (en) * | 2011-03-15 | 2012-09-19 | 中国科学院沈阳应用生态研究所 | Method for extracting total DNA (deoxyribonucleic acid) of litter |
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| CN102676496A (en) * | 2011-03-15 | 2012-09-19 | 中国科学院沈阳应用生态研究所 | Method for extracting total DNA (deoxyribonucleic acid) of litter |
Non-Patent Citations (3)
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| 一种从果树成熟叶片提取DNA 的方法;佟兆国等;《果树学报》;20080229;第25卷(第1期);第122-125页 * |
| 沈晓岚等.观赏凤梨DNA 提取方法研究.《北方园艺》.2009,(第12期),第114-117页. * |
| 火龙果总DNA提取方法比较研究;余志雄等;《中国农学通报》;20100228;第26卷(第4期);第301页第1.3.2节 * |
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