CN104152435A - High-efficiency extraction method for trace cherry leaf DNA (deoxyribonucleic acid) - Google Patents
High-efficiency extraction method for trace cherry leaf DNA (deoxyribonucleic acid) Download PDFInfo
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- CN104152435A CN104152435A CN201410360484.0A CN201410360484A CN104152435A CN 104152435 A CN104152435 A CN 104152435A CN 201410360484 A CN201410360484 A CN 201410360484A CN 104152435 A CN104152435 A CN 104152435A
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Abstract
The invention discloses a high-efficiency extraction method for trace cherry leaf DNA (deoxyribonucleic acid), belonging to the technical field of molecular biology. The method comprises the following steps: taking trace cherry leaf which is sufficiently dried by allochroic silicagel, putting in a 2ml centrifuge tube, quickly mashing the leaf into powder by using a glass bar with a sanded tail end and with the diameter of 0.6cm, washing by using a buffer solution, cracking, extracting, precipitating, detecting and the like to finally obtaining the high-concentration high-purity DNA. The method is substituted for liquid nitrogen grinding which is commonly used in the traditional DNA extraction method. The method can still obtain high-quality DNA under the condition of no using liquid nitrogen, and can prevent the material from browning in the liquid nitrogen grinding process. The method is simple to operate, is practical and convenient, has high efficiency, and has important functions for DNA extraction of trace rare materials.
Description
Technical field
The invention belongs to technical field of molecular biology, be specially a kind of micro-sweet cherry leaves DNA highly effective extraction method that is applicable to.
Background technology
Obtain high quality (high density, high purity, high integrality) DNA be molecular biology experiment the most basic be also the first step of most critical, at present, develop several different methods, also successfully from the histoorgans such as plant leaf, callus, tissue cultured seedling, fruit, phloem, extracted DNA.But for some scarce resources or material, micro-material as remaining in plant, the research material such as seedling, the early stage plant of xylophyta first familiar generation nursery stock that in molecular mark, offspring is born soon all cannot obtain the extraction of a large amount of plant tissue materials for DNA, and cause Efficiency lower, research cycle is long, but also easily causes the dead of research material and lose.
Cherry is typical perennial woody fruit tree crop, the growth velocity of its first familiar generation is extremely slow, and the seed of gathering then generally will arrive the Second Year seedling of could growing, these biological characteristicses have greatly hindered the early stage acquisition of its mapping population DNA in Genetic linkage map builds, and the molecular biology of some breeding correlated character identifies in early days, and delay flow of research.The most important thing is that some seedling is easily dead in process of growth and cause the loss of research material.Therefore how significant for the molecular biology research of most of materials at the high-quality DAN of the early stage acquisition of plant.
Summary of the invention
Few and cannot obtain high-quality genomic dna for follow-up study at seedling leaf and other organization material in order to solve cherry.The invention provides a kind of micro-sweet cherry leaves DNA highly effective extraction method that is applicable to.Adopt method of the present invention in cherry Seedling Stage or remaining micro-blade, to obtain more high quality genomic dna, but also omit the liquid nitrogen grinding using in traditional method, reduce extraction cost, the less oxidation of organization material in leaching process.The method is simple to operate, practical and convenient, and efficiency is high, especially has vital role for the DNA extraction of the precious material of trace.
Its technical scheme is:
A kind of applicable micro-sweet cherry leaves DNA highly effective extraction method, comprises the following steps:
1) material is prepared: gather fresh cherry spire sheet and be placed in sealed bag, according to 1: 20-1: 50 ratio adds discolour silica gel, and fully mixes, until blade complete drying.
2) get fully dry sweet cherry leaves 0.01-0.03g and be placed in 2ml centrifuge tube, the glass stick that is polished into level and smooth ovum head with diameter 0.6cm leading portion is smashed dry blade to pieces fast to being Powdered.
3) add 1ml damping fluid 1 and 10 μ l beta-mercaptoethanols, after fully mixing, at the centrifugal 3min of 5000rpm at normal temperatures, abandon supernatant, collecting precipitation.
4) in precipitation, add 3% CTAB damping fluid and 10 μ l beta-mercaptoethanols of 1ml65 DEG C of preheating, 65 DEG C of water-bath 1h, put upside down centrifuge tube so that liquid blending every 5-10min during this time, then the centrifugal 10min of 12500rpm at 26 DEG C;
5) get in the centrifuge tube that supernatant liquor proceeds to new 2.0ml, add isopyknic chloroform, jog extraction, the centrifugal 10min of 12500rpm at 5 DEG C;
6) supernatant liquor proceeds in new 2.0ml centrifuge tube, repeating step 5);
7) get supernatant liquor approximately 600 μ l and proceed in new 1.5ml centrifuge tube, add the sodium-acetate of 2 times of volumes through the dehydrated alcohol of-20 DEG C of precoolings and the 3mol/L of 1/10 volume, put upside down and mix-20 placement 30min, the centrifugal 15min of 11000rpm, abandons supernatant, collecting precipitation;
8) in DNA precipitation, add 75% ethanol 800 μ l, jog is also placed 5min, outwells ethanol, so cleans twice, then cleans once with dehydrated alcohol, is placed in ventilation and spends the night and dry up;
9) 50 μ l TE dissolvings for DNA precipitation, then add the RNase A of 1 μ l10ng/ μ l, normal temperature to spend the night to be placed on-20 DEG C of Refrigerator stores for subsequent use;
10) get the DNA solution that 2 μ l dissolve, detect through 1.0% agarose gel electrophoresis, and detect 260nm, the absorption value at 280nm place and the concentration of DNA on Eppendorf Biophotomete type nucleic acid protein analyser, and judge the purity of DNA according to OD 260/OD280 value.
Step 1) described in the fresh blade of cherry be the blade without disease and pest, and with young leaflet tablet the best, can be contained in the good tea bag of permeability and then be placed in the discolour silica gel that point pocket adds corresponding proportion for less blade material, drying material should be handled with care, prevents that dry blade from cannot separate after cracked from discolour silica gel.
Step 2) described in glass stick should polish in advance, its front end or two need be polished into level and smooth ovum head, to better smash blade to pieces.
Step 3) described in damping fluid 1 be 1mol/L sodium-chlor; 0.1mol/L Tris-HCl PH8.0; 0.02mol/L EDTA PH8.0; 0.4mol/L glucose.
Step 4) described in CTAB damping fluid be 1.5mol/lNaCl; 0.1mol/L Tris-Hcl PH8.0; 0.02mol/L EDTA PH8.0; 2%PVP; 3%CTAB.
Step 4) in should fully shake up in time after adding CTAB lysate, be as much as possible suspension, in water-bath process, every the upper and lower jog of 3-5min once, ensure that cracking is abundant.
Step 7) in precipitation all dehydrated alcohols are minimum will add 2 times of volumes, can effectively remove color if the darker words of precipitated liquid color are added sodium-acetate (20-100ul) in right amount.
Step 8) in 75% ethanol of washing precipitation at least want 800 μ l, washing times can according to circumstances increase, and finally dries up with absolute ethanol washing.
Compared with prior art, beneficial effect of the present invention is:
The present invention is intended to overcome and cannot obtains enough organization materials and cannot extract the difficult problem of genomic dna for the growth of seedling initial stage.Adopt extracting method flow process of the present invention can be from micro-blade material (about 0.01g) high efficiency extraction genomic dna.High (the OD260/OD280=1.77-1.94 of DNA purity obtaining; OD260/OD230=1.95-2.11), integrity and good fluidity (can be dissolved in rapidly water or TE, can not separate out precipitation after dissolving, solution is thickness not, and 1.0% agarose gel electrophoresis detects fragment and is greater than 20Kb).
1, the method for the invention is to be dried blade as extracting material, compared to fresh or freezing young leaflet tablet for tradition, the method is loose to the requirement of extraction material, can be suitable for the extraction of field or remote material, and avoided young tender material oxidized and browning in liquid nitrogen grinding process, the genomic dna of acquisition can meet subsequent experimental requirement.
2, the inventive method has substituted original liquid nitrogen grinding method on leaf tissue breaking method, can be in the situation that only having micro-blade material direct crushing material in centrifuge tube, the breaking method of leaf tissue while having simplified DNA extraction, avoid using liquid nitrogen, can realize again and the in the situation that of micro-blade material, extract efficiently genomic dna.Both can save blade material with respect to traditional extracting method, short form test step, can reduce again extraction cost.
3, adopt method of the present invention can shorten extraction time to 2.5 hour, experiment flow is simplified relatively.
Brief description of the drawings
Fig. 1 is the total genomic dna with the cherry of method extraction of the present invention, through 1.0% agarose gel electrophoresis detected result, note: sepharose concentration is 1.0%, M1 is Markerl, fragment is followed successively by 546bp, 2027bp, 2322bp, 4361bp, 9416bp, 23130bp from small to large, M2 is Markerl, and fragment is followed successively by 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp from small to large.
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described further.
A kind of applicable micro-sweet cherry leaves DNA highly effective extraction method, comprises the following steps:
1) in the wild or to gather respectively fresh cherry (Luoyang, henan), wild Chinese cherry (Beichuan, Sichuan), sweet cherry (early beautiful in field, Hanyuan County, sichuan Province) and the each 0.5-1.0g of Nanking cherry (Pingliang, Gansu) blade, spire sheet is placed in sealed bag, add 10-50g discolour silica gel, and fully mixing, until blade complete drying.
2) get fully dry sweet cherry leaves 0.01-0.03g and be placed in 2ml centrifuge tube, the glass stick that is polished into level and smooth ovum head with diameter 0.6cm leading portion is smashed dry blade to pieces fast to being Powdered.
3) add 1ml damping fluid 1 and 10 μ l beta-mercaptoethanols, after fully mixing, at the centrifugal 3min of 5000rpm at normal temperatures, abandon supernatant, collecting precipitation.
4) in precipitation, add 3% CTAB damping fluid and 10 μ l beta-mercaptoethanols of 1ml65 DEG C of preheating, 65 DEG C of water-bath 1h, put upside down centrifuge tube so that liquid blending every 5-10min during this time, then the centrifugal 10min of 12500rpm at 26 DEG C;
5) get in the centrifuge tube that supernatant liquor proceeds to new 2.0ml, add isopyknic chloroform, jog extraction, the centrifugal 10min of 12500rpm at 5 DEG C;
6) supernatant liquor proceeds in new 2.0ml centrifuge tube, repeating step 5);
7) get supernatant liquor approximately 600 μ l and proceed in new 1.5ml centrifuge tube, add the sodium-acetate of 2 times of volumes through the dehydrated alcohol of-20 DEG C of precoolings and the 3mol/L of 1/10 volume, put upside down and mix-20 placement 30min, the centrifugal 15min of 11000rpm, abandons supernatant, collecting precipitation;
8) in DNA precipitation, add 75% ethanol 800 μ l, jog is also placed 5min, outwells ethanol, so cleans twice, then cleans once with dehydrated alcohol, is placed in ventilation and spends the night and dry up;
9) 50 μ l TE dissolvings for DNA precipitation, then add the RNase A of 1 μ l10ng/ μ l, normal temperature to spend the night to be placed on-20 DEG C of Refrigerator stores for subsequent use;
10) get the DNA solution that 2 μ l dissolve, detect (Fig. 1 is followed successively by each 2 of cherry, sweet cherry, wild Chinese cherry, Nanking cherry from left to right) through 1.0% agarose gel electrophoresis.
11) on Eppendorf Biophotomete type nucleic acid protein analyser, detect 260nm, the absorption value at 280nm place and the concentration of DNA after getting 50 times of DNA solution dilutions that 2 μ l dissolve, and judge the purity of DNA according to OD 260/OD 280 and OD 260/OD230 value.
The DNA detection information that table 1 extracts for the leaf quality of examination material
| ? | 49 | 50 | 51 | 52 | 53 | 54 | 55 | 56 |
| Leaf quality (g) | 0.0126 | 0.0274 | 0.0217 | 0.0175 | 0.0087 | 0.0108 | 0.0133 | 0.0164 |
| (ng/ μ l) for concentration | 150 | 151.4 | 90.7 | 180 | 87.1 | 119.1 | 130.6 | 56.7 |
| OD?260/OD?280 | 1.86 | 1.84 | 1.77 | 1.82 | 1.84 | 1.91 | 1.89 | 1.94 |
| OD?260/OD?230 | 2.08 | 2.11 | 2.03 | 1.98 | 2.17 | 1.95 | 1.99 | 2.07 |
The above; it is only preferably embodiment of the present invention; protection scope of the present invention is not limited to this; anyly be familiar with those skilled in the art; in the technical scope disclosing in the present invention, the simple transformation of the technical scheme that can obtain apparently or equivalence are replaced and are all fallen within the scope of protection of the present invention.
Claims (5)
1. be applicable to a micro-sweet cherry leaves DNA highly effective extraction method, it is characterized in that, comprise the following steps:
1) material is prepared: gather fresh cherry spire sheet and be placed in sealed bag, according to 1: 20-1: 50 ratio adds discolour silica gel, and fully mixes, until blade complete drying;
2) get fully dry sweet cherry leaves 0.01-0.03g and be placed in 2ml centrifuge tube, the glass stick that is polished into level and smooth ovum head with diameter 0.6cm leading portion is smashed dry blade to pieces fast to being Powdered;
3) add 1ml damping fluid 1 and 10 μ l beta-mercaptoethanols, after fully mixing, at the centrifugal 3min of 5000rpm at normal temperatures, abandon supernatant, collecting precipitation;
4) in precipitation, add 3% CTAB damping fluid and 10 μ l beta-mercaptoethanols of 1ml65 DEG C of preheating, 65 DEG C of water-bath 1h, put upside down centrifuge tube so that liquid blending every 5-10min during this time, then the centrifugal 10min of 12500rpm at 26 DEG C;
5) get in the centrifuge tube that supernatant liquor proceeds to new 2.0ml, add isopyknic chloroform, jog extraction, the centrifugal 10min of 12500rpm at 5 DEG C;
6) supernatant liquor proceeds in new 2.0ml centrifuge tube, repeating step 5);
7) get supernatant liquor approximately 600 μ l and proceed in new 1.5ml centrifuge tube, add the sodium-acetate of 2 times of volumes through the dehydrated alcohol of-20 DEG C of precoolings and the 3mol/L of 1/10 volume, put upside down and mix-20 placement 30min, the centrifugal 15min of 11000rpm, abandons supernatant, collecting precipitation;
8) in DNA precipitation, add 75% ethanol 800 μ l, jog is also placed 5min, outwells ethanol, so cleans twice, then cleans once with dehydrated alcohol, is placed in ventilation and spends the night and dry up;
9) 50 μ l TE dissolvings for DNA precipitation, then add the RNase A of 1 μ l10ng/ μ l, normal temperature to spend the night to be placed on-20 DEG C of Refrigerator stores for subsequent use;
10) get the DNA solution that 2 μ l dissolve, detect through 1.0% agarose gel electrophoresis, and detect 260nm, the absorption value at 280nm place and the concentration of DNA on Eppendorf Biophotomete type nucleic acid protein analyser, and judge the purity of DNA according to OD 260/OD280 value.
2. one according to claim 1 is applicable to micro-sweet cherry leaves DNA highly effective extraction method, it is characterized in that, step 1) described in the fresh blade of cherry be the blade without disease and pest, and with young leaflet tablet the best, can be contained in the good tea bag of permeability and then be placed in the discolour silica gel that point pocket adds corresponding proportion for less blade material, drying material should be handled with care, prevents that dry blade from cannot separate after cracked from discolour silica gel.
One according to claim 1 be applicable to micro-sweet cherry leaves DNA highly effective extraction method, it is characterized in that step 2) described in glass stick should polish in advance, its front end or two need be polished into level and smooth ovum head, to better smash blade to pieces.
One according to claim 1 be applicable to micro-sweet cherry leaves DNA highly effective extraction method, it is characterized in that step 3) described in damping fluid 1 be 1mol/L sodium-chlor; 0.1mol/L Tris-HCl PH8.0; 0.02mol/L EDTA PH8.0; 0.4mol/L glucose.
One according to claim 1 be applicable to micro-sweet cherry leaves DNA highly effective extraction method, it is characterized in that step 4) described in CTAB damping fluid be 1.5mol/lNaCl; 0.1mol/L Tris-Hcl PH8.0; 0.02mol/L EDTA PH8.0; 2%PVP; 3%CTAB.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104371998A (en) * | 2014-11-25 | 2015-02-25 | 福建省农业科学院甘蔗研究所 | DNA extracting method for bitter gourd hybrid purity molecular identification |
| CN105758692A (en) * | 2016-04-29 | 2016-07-13 | 中国科学院西北高原生物研究所 | Macro fungus pulverizing method suitable for laboratory use |
| CN109880822A (en) * | 2019-03-12 | 2019-06-14 | 中国林业科学研究院亚热带林业研究所 | A kind of idesia high quality DNA extracting method |
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| CN102337330A (en) * | 2011-07-18 | 2012-02-01 | 北京振东光明药物研究院有限公司 | Method for identifying DNA (Deoxyribose Nucleic Acid) molecule in white glabrous greenbrier rhizome original plant |
| CN103756994A (en) * | 2013-03-21 | 2014-04-30 | 四川农业大学 | DNA extraction method for polysaccharide-rich plant dried leaves |
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2014
- 2014-07-24 CN CN201410360484.0A patent/CN104152435A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102337330A (en) * | 2011-07-18 | 2012-02-01 | 北京振东光明药物研究院有限公司 | Method for identifying DNA (Deoxyribose Nucleic Acid) molecule in white glabrous greenbrier rhizome original plant |
| CN103756994A (en) * | 2013-03-21 | 2014-04-30 | 四川农业大学 | DNA extraction method for polysaccharide-rich plant dried leaves |
Non-Patent Citations (1)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104371998A (en) * | 2014-11-25 | 2015-02-25 | 福建省农业科学院甘蔗研究所 | DNA extracting method for bitter gourd hybrid purity molecular identification |
| CN105758692A (en) * | 2016-04-29 | 2016-07-13 | 中国科学院西北高原生物研究所 | Macro fungus pulverizing method suitable for laboratory use |
| CN109880822A (en) * | 2019-03-12 | 2019-06-14 | 中国林业科学研究院亚热带林业研究所 | A kind of idesia high quality DNA extracting method |
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