CN104928281A - Method for improving DNA (Deoxyribonucleic Acid) extraction quality of cotton seed - Google Patents
Method for improving DNA (Deoxyribonucleic Acid) extraction quality of cotton seed Download PDFInfo
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Abstract
The invention relates to a DNA (Deoxyribonucleic Acid) extraction method, in particular to a method for improving the DNA extraction quality of a cotton seed. 1 percent of Tween-20 is added into an extraction buffer solution obtained through a CTAB (Cetyl Trimethyl Ammonium Bromide) method or an SDS (Sodium Dodecyl Sulfate) method, and then the DNA of the cotton seed is extracted. According to the method, 1 percent of the Tween-20 surfactant is added into a CTAB extraction buffer solution and an SDS extraction buffer solution, and the extraction buffer solutions are in more complete contact with experimental materials of the cotton seed, so that more impurities are removed, and the OD260/280 value of the obtained DNA is increased.
Description
Technical field
The present invention relates to DNA extraction method, specifically, relate to a kind of method improving cotton seeds DNA extraction quality.
Background technology
Extracting genome DNA is the basis of molecular biology research.Conventional DNA extraction method mainly contains cetyltriethylammonium bromide (CTAB) method, sodium lauryl sulphate (SDS) method.SDS method is simple to operate, but products therefrom is impure more; CTAB can fracturing cell walls and cytolemma, and can form mixture with nucleic acid, and then isolates high quality genomic dna from the plant tissue being rich in polyphenol and polysaccharide.The content difference of the material composition contained by differing materials and various composition is comparatively large, therefore, extracts should take diverse ways for different plant genome DNA.
Due to secondary metabolite impurity such as cotton seeds rich in proteins, gossypol, polysaccharide, tannin, traditional CTAB method and the extraction effect of SDS method to the seed being rich in impurity not good, gained DNA purity is very low.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide a kind of method improving cotton seeds DNA extraction quality.
Improve a method for cotton seeds DNA extraction quality, it is carry out cotton seeds DNA extraction add 1%Tween-20 (i.e. polysorbas20) (V/V) in the Extraction buffer of CTAB method or SDS method after.Namely the CTAB Extraction buffer replaced in CTAB method with CTAB-1%Tween20 Extraction buffer carries out cotton seeds DNA extraction, or carries out cotton seeds DNA extraction with the SDS Extraction buffer that SDS-1%Tween20 Extraction buffer is replaced in SDS method.
Wherein, described CTAB-1%Tween20 Extraction buffer is: 100mmol/LTris-HCl, 20mmol/L EDTA, 2%CTAB (W/V), 2%PVP40 (W/V), 8%NaCl (W/V), 2% beta-mercaptoethanol (W/V), 1%Tween-20 (V/V).
Wherein, described SDS-1%Tween20 Extraction buffer is: 1%SDS (W/V), 10mmol/L EDTA, 8%NaCl (W/V), 50mmol/L Tris-HCl, 0.5% sorbyl alcohol (W/V), 1%PVP40 (W/V), 2% beta-mercaptoethanol (W/V), 1%Tween-20 (V/V).
The concrete steps of carrying out cotton seeds DNA extraction with the CTAB-1%Tween20 Extraction buffer CTAB Extraction buffer replaced in CTAB method are as follows:
1) 30Times/s after load weighted cotton seeds lyophilize 24h is ground 35s, add the 800 μ l CTAB-1%Tween20 Extraction buffers through 65 DEG C of preheatings, concussion 30s, 65 DEG C of water-baths 40 minutes;
2) add the chloroform of 800 μ l: the volume ratio of primary isoamyl alcohol is the solution of 24:1, mix 10 minutes, centrifugal 10 minutes of 10000r/min; Get supernatant liquor and add isopyknic chloroform: the volume ratio of primary isoamyl alcohol is that the solution of 24:1 is brought up again once, centrifugal 10 minutes of 10000r/min;
3) get supernatant liquor, add the dehydrated alcohol of 2 times of volumes through-20 DEG C of precoolings, slowly put upside down more than 15 times, in-20 DEG C of ice baths more than 30 minutes, then centrifugal 7 minutes of 12000r/min; Incline supernatant liquor, washes 2 times with the ethanol of volumn concentration 70%, and dehydrated alcohol is washed once, adds 200 μ l TE damping fluids, 4 DEG C of preservations after air-dry 30 minutes to drying.
The concrete steps of carrying out cotton seeds DNA extraction with the SDS-1%Tween20 Extraction buffer SDS Extraction buffer replaced in SDS method are as follows:
1) 30Times/s after load weighted cotton seeds lyophilize 24h is ground 35s, add the 800 μ l SDS-1%Tween20 Extraction buffers through 65 DEG C of preheatings, concussion 30s, 65 DEG C of water-baths 40 minutes;
2) add the chloroform of 800 μ l: the volume ratio of primary isoamyl alcohol is the solution of 24:1, mix 10 minutes, centrifugal 10 minutes of 10000r/min; Get supernatant liquor and add isopyknic chloroform: the volume ratio of primary isoamyl alcohol is that the solution of 24:1 is brought up again once, centrifugal 10 minutes of 10000r/min;
3) supernatant liquor is got, add the Virahol of 2 times of volumes through-20 DEG C of precoolings, slowly put upside down more than 15 times, in-20 DEG C of ice baths more than 30 minutes, then centrifugal 7 minutes of 12000r/min, incline supernatant liquor, wash 2-3 time with the ethanol of volumn concentration 70%, dehydrated alcohol is washed once, adds 200 μ l TE damping fluids, 4 DEG C of preservations after air-dry 30 minutes to drying.
The present invention also provides CTAB-1%Tween20 Extraction buffer, it is: 100mmol/LTris-HCl, 20mmol/L EDTA, 2%CTAB (W/V), 2%PVP40 (W/V), 8%NaCl (W/V), 2% beta-mercaptoethanol (W/V), 1%Tween-20 (V/V).
The present invention also provides SDS-1%Tween20 Extraction buffer, it is: 1%SDS (W/V), 10mmol/L EDTA, 8%NaCl (W/V), 50mmol/L Tris-HCl, 0.5% sorbyl alcohol (W/V), 1%PVP40 (W/V), 2% beta-mercaptoethanol (W/V), 1%Tween-20 (V/V).
The method that the present invention further provides described raising cotton seeds DNA extraction quality is extracting the application in cotton seeds DNA.
The present invention further provides described CTAB-1%Tween20 Extraction buffer and the application of goods in extraction cotton seeds DNA thereof.
The present invention further provides described SDS-1%Tween20 Extraction buffer and the application in extraction cotton seeds DNA thereof.
Beneficial effect of the present invention is:
The present invention, by adding 1%Tween-20 tensio-active agent in CTAB Extraction buffer and SDS Extraction buffer, makes the contact of Extraction buffer and cotton seeds experiment material more abundant, thus removes more impurity, improve the OD of gained DNA
260/280value.
Accompanying drawing explanation
The agarose gel electrophoresis detection figure of the cotton seeds DNA that Fig. 1 is the embodiment of the present invention 1, embodiment 2, comparative example 1, comparative example 2, comparative example 3, comparative example 4 method are extracted.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
1, selection is tested:
The seed of transgenic pest-resistant phytocide cotton (CCRI provides), non-transgenic cotton stone 321 seeds far away.
2, DNA extraction damping fluid and composition thereof:
CTAB Extraction buffer: 100mmol/L Tris-HCl, 20mmol/L EDTA, 2%CTAB (W/V), 2%PVP40 (W/V), 8%NaCl (W/V), 2% beta-mercaptoethanol (W/V).
CTAB-0.5%Tween20 Extraction buffer: 100mmol/L Tris-HCl, 20mmol/L EDTA, 2%CTAB (W/V), 2%PVP40 (W/V), 8%NaCl (W/V), 2% beta-mercaptoethanol (W/V), 0.5%Tween-20 (V/V).
CTAB-1%Tween20 Extraction buffer: 100mmol/L Tris-HCl, 20mmol/LEDTA, 2%CTAB (W/V), 2%PVP40 (W/V), 8%NaCl (W/V), 2% beta-mercaptoethanol (W/V), 1%Tween-20 (V/V).
SDS Extraction buffer: 1%SDS (W/V), 10mmol/L EDTA, 8%NaCl (W/V), 50mmol/L Tris-HCl, 0.5% sorbyl alcohol (W/V), 1%PVP40 (W/V), 2% beta-mercaptoethanol (W/V).
SDS-0.5%Tween20 Extraction buffer: 1%SDS (W/V), 10mmol/LEDTA, 8%NaCl (W/V), 50mmol/L Tris-HCl, 0.5% sorbyl alcohol (W/V), 1%PVP40 (W/V), 2% beta-mercaptoethanol (W/V), 0.5%Tween-20 (V/V).
SDS-1%Tween20 Extraction buffer: 1%SDS (W/V), 10mmol/L EDTA, 8%NaCl (W/V), 50mmol/L Tris-HCl, 0.5% sorbyl alcohol (W/V), 1%PVP40 (W/V), 2% beta-mercaptoethanol (W/V), 1%Tween-20 (V/V).
TE damping fluid: 10mmol/L Tris-HCl, 1mmol/L EDTA
Other reagent: chloroform: primary isoamyl alcohol (24:1) is chloroform: the volume ratio of primary isoamyl alcohol is the solution of 24:1,70% ethanol (V/V), dehydrated alcohol, Virahol.
Embodiment 1 CTAB-1%Tween20 method extracts cotton seeds DNA
Method with reference to Sun Guoli (Song Guoli, 1998) is also changed a little, and operation steps is as follows:
Be placed in bottom 2ml centrifuge tube by load weighted cotton seeds 19.36mg, add little steel ball, open tube lid puts into freeze drier, puts into rapidly vibration grinding 35s (30Times/s) in ball milling instrument after lyophilize 24h.Then add the 800 μ lCTAB-1%Tween20 Extraction buffers through 65 DEG C of preheatings, be placed at about 30s on oscillator, put into 65 DEG C of water-baths 40 minutes, turn upside down 1 time every 10min.The chloroform of 800 μ l is added: primary isoamyl alcohol (24:1), upper and lower jog mixing about 10 minutes, centrifugal 10 minutes of 10000r/min after water-bath.Get supernatant liquor (about 600 μ l) and, in 1.5ml centrifuge tube, add isopyknic chloroform: primary isoamyl alcohol (24:1) is brought up again once, centrifugal 10 minutes of 10000r/min.Refetch in supernatant liquor (about 500 μ l) to new 1.5ml centrifuge tube, add warp-20 DEG C of precooling dehydrated alcohols of 2 times of volumes (about 1ml), slowly put upside down centrifuge tube more than 15 times.More than 30 minutes are left standstill, then centrifugal 7 minutes of 12000r/min in-20 DEG C of ice baths.Carefully incline supernatant liquor, washes 2 times with 70% ethanol, and dehydrated alcohol is washed once, adds 200 μ l TE after air-dry 30 minutes to drying, 4 DEG C of preservations.
Embodiment 2 SDS-1%Tween20 method extracts cotton seeds DNA
Reference Guo Baosheng (rectify violent, 2010; Guo Baosheng, 2005; Liu Feng, 2010) method is also changed a little, and operation steps is as follows:
Be placed in bottom 2ml centrifuge tube by load weighted cotton seeds 19.36mg, add little steel ball, open tube lid puts into freeze drier, puts into rapidly vibration grinding 35s (30Times/s) in ball milling instrument after lyophilize 24h.Then add the 800 μ lSDS-1%Tween20 Extraction buffers through 65 DEG C of preheatings, be placed at about 30s on oscillator, put into 65 DEG C of water-baths 40 minutes, turn upside down 1 time every 10min.The chloroform of 800 μ l is added: primary isoamyl alcohol (24:1), upper and lower jog mixing about 10 minutes, centrifugal 10 minutes of 10000r/min after water-bath.Get supernatant liquor (about 600 μ l) and, in 1.5ml centrifuge tube, add isopyknic chloroform: primary isoamyl alcohol (24:1) is brought up again once, centrifugal 10 minutes of 10000r/min.Refetch in supernatant liquor (about 500 μ l) to new 1.5ml centrifuge tube, add the pre-cold isopropanol of-20 DEG C, warp of 2 times of volumes (about 1ml), slowly put upside down centrifuge tube more than 15 times.More than 30 minutes are left standstill, then centrifugal 7 minutes of 12000r/min in-20 DEG C of ice baths.Carefully incline supernatant liquor, washes 2-3 time with 70% ethanol, and dehydrated alcohol is washed once, adds 200 μ l TE after air-dry 30 minutes to drying, 4 DEG C of preservations.
Comparative example 1 CTAB method extracts cotton seeds DNA
Replace CTAB-1%Tween20 Extraction buffer with CTAB Extraction buffer, other step is with embodiment 1.
Comparative example 2 CTAB-0.5%Tween20 method extracts cotton seeds DNA
Replace CTAB-1%Tween20 Extraction buffer with CTAB-0.5%Tween20 Extraction buffer, other step is with embodiment 1.
Comparative example 3 SDS method extracts cotton seeds DNA
Replace SDS-1%Tween20 Extraction buffer with SDS Extraction buffer, other step is with embodiment 2.
Comparative example 4 SDS-0.5%Tween20 method extracts cotton seeds DNA
Replace SDS-1%Tween20 Extraction buffer with SDS-0.5%Tween20 Extraction buffer, other step is with embodiment 2.
The quality examination of experimental result DNA
1, ultraviolet spectrophotometry detects
Using TE as blank, utilize micro-ultraviolet spectrophotometer (Nanodrop2000, Thermo Scientific) detect embodiment 1-2 and comparative example 1-4 extract concentration, the OD of cotton DNA
260/280value, and according to the Mass Calculation yield weighed before extraction DNA.Wherein OD
260/280value should be suitable in 1.8 ~ 2.2 scopes.
2, PCR and electrophoresis detection
(1) DNA profiling dilution
Because PCR uses DNA profiling 2 μ l, need to ensure that the content of DNA in PCR reaction system is between 20ng ~ 50ng simultaneously, therefore need to be diluted to 30ng/ μ l with TE to DNA sample is unified, be positioned over preserve in-20 DEG C of refrigerators stand-by.
(2) pcr amplification of DNA sample and electrophoresis detection
Because selected cotton material is all transgenic pest-resistant phytocide cotton kind, therefore Auele Specific Primer is selected to carry out PCR qualitative detection to Cry I A (c) gene in sample.Upstream and downstream primer is respectively 5 ' GAAGGATTGAGCAATCTCTAC 3 ' and 5 ' CAATCAGCCTAGTAAGGTCGT 3 '.PCR reaction system: 2 × Taq PCRMaster Mix10 μ l (the strong Xin Borui Bioisystech Co., Ltd in Beijing), the each 0.5 μ l of upstream and downstream primer (10u/mol) (Shanghai Sheng Gong Bioisystech Co., Ltd), DNA profiling 2 μ l, ddH
2o 7 μ l.
Response procedures adopts: denaturation 95 DEG C of 4min, sex change 94 DEG C of 1min, annealing 56 DEG C of 1min, extension 72 DEG C of 1.5min, 30 circulations, total elongation 72 DEG C of 5min after loop ends, 4 DEG C of preservations.The object fragment length of amplification gene Cry I A (c) is approximately 340bp, uses the agarose gel electrophoresis of 2% to detect.
3, interpretation of result:
Table 1 CTAB method, CTAB-0.5%Tween20 method extract comparing of seed DNA mass discrepancy with CTAB-1%Tween20 method
For seed CTAB method, CTAB-0.5%Tween20 method and CTAB-1%Tween20 method, CTAB-0.5%Tween20 method and CTAB-1%Tween20 method gained DNA concentration and yield are significantly lower than CTAB method, but because CTAB-0.5%Tween20 method and CTAB-1%Tween20 method gained DNA concentration are on average all at 228ng/ more than μ l, meet the requirements, be enough to carry out subsequent experimental.The OD of CTAB-1%Tween20 method gained DNA
260/280value is significantly higher than CTAB-0.5%Tween20 method and CTAB method, and its mean value is between 1.8 to 2.2, and comparatively CTAB-0.5%Tween20 method and CTAB method are significantly increased the purity of Gu CTAB-1%Tween20 method gained DNA.Also can find out from the electrophorogram (see Fig. 1) of these three kinds of methods, the electrophorogram sharpness of CTAB-1%Tween20 method gained DNA compared with CTAB-0.5%Tween20 method and CTAB method clear.
It can thus be appreciated that CTAB-1%Tween20 method also can obtain enough DNA and carry out subsequent experimental, comparatively CTAB-0.5%Tween20 method and CTAB method have a significant improvement for its gained DNA purity and electrophorogram sharpness simultaneously.
Table 2 SDS method, SDS-0.5%Tween20 method extract comparing of seed DNA mass discrepancy with SDS-1%Tween20 method
For seed SDS method, SDS-0.5%Tween20 method and SDS-1%Tween20 method, SDS-0.5%Tween20 method and SDS-1%Tween20 method gained DNA concentration and yield are significantly lower than SDS method, but because SDS-0.5%Tween20 method and SDS-1%Tween20 method gained DNA concentration are on average all at 369ng/ more than μ l, therefore meet the requirements, be enough to carry out subsequent experimental.The OD of SDS-1%Tween20 method gained DNA
260/280value is significantly higher than SDS-0.5%Tween20 method and SDS method, and comparatively SDS-0.5%Tween20 method and SDS method are significantly increased the purity of Gu SDS-1%Tween20 method gained DNA.Can find out from the electrophorogram (see Fig. 1) of these two kinds of methods, comparatively SDS-0.5%Tween20 method and SDS method do not have marked difference to the electrophorogram sharpness of SDS-1%Tween20 method gained DNA yet.
It can thus be appreciated that, SDS-1%Tween20 method also can obtain enough DNA and carry out subsequent experimental, and its electrophorogram sharpness and SDS-0.5%Tween20 method, SDS method do not have significant difference, comparatively SDS-0.5%Tween20 method and SDS method have a significant improvement its gained DNA purity simultaneously.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. improve a method for cotton seeds DNA extraction quality, it is characterized in that, add 1%Tween20 (V/V) in the Extraction buffer of CTAB method or SDS method after, carry out cotton seeds DNA extraction.
2. the method for raising cotton seeds DNA extraction quality according to claim 1, it is characterized in that, cotton seeds DNA extraction is carried out with the CTAB Extraction buffer that CTAB-1%Tween20 Extraction buffer is replaced in CTAB method, described CTAB-1%Tween20 Extraction buffer is: 100mmol/L Tris-HCl, 20mmol/L EDTA, 2%CTAB (W/V), 2%PVP40 (W/V), 8%NaCl (W/V), 2% beta-mercaptoethanol (W/V), 1%Tween-20 (V/V).
3. the method for raising cotton seeds DNA extraction quality according to claim 2, it is characterized in that, concrete steps are as follows:
1) 30Times/s after load weighted cotton seeds lyophilize 24h is ground 35s, add the 800 μ l CTAB-1%Tween20 Extraction buffers through 65 DEG C of preheatings, concussion 30s, 65 DEG C of water-baths 40 minutes;
2) add the chloroform of 800 μ l: the volume ratio of primary isoamyl alcohol is the solution of 24:1, mix 10 minutes, centrifugal 10 minutes of 10000r/min; Get supernatant liquor and add isopyknic chloroform: the volume ratio of primary isoamyl alcohol is that the solution of 24:1 is brought up again once, centrifugal 10 minutes of 10000r/min;
3) get supernatant liquor, add the dehydrated alcohol of 2 times of volumes through-20 DEG C of precoolings, slowly put upside down more than 15 times, in-20 DEG C of ice baths more than 30 minutes, then centrifugal 7 minutes of 12000r/min; Incline supernatant liquor, washes 2 times with the ethanol of volumn concentration 70%, and dehydrated alcohol is washed once, adds 200 μ l TE damping fluids, 4 DEG C of preservations after air-dry 30 minutes to drying.
4. the method for raising cotton seeds DNA extraction quality according to claim 1, it is characterized in that, cotton seeds DNA extraction is carried out with the SDS Extraction buffer that SDS-1%Tween20 Extraction buffer is replaced in SDS method, described SDS-1%Tween20 Extraction buffer is: 1%SDS (W/V), 10mmol/L EDTA, 8%NaCl (W/V), 50mmol/L Tris-HCl, 0.5% sorbyl alcohol (W/V), 1%PVP40 (W/V), 2% beta-mercaptoethanol (W/V), 1%Tween-20 (V/V).
5. the method for raising cotton seeds DNA extraction quality according to claim 4, it is characterized in that, concrete steps are as follows:
1) 30Times/s after load weighted cotton seeds lyophilize 24h is ground 35s, add the 800 μ l SDS-1%Tween20 Extraction buffers through 65 DEG C of preheatings, concussion 30s, 65 DEG C of water-baths 40 minutes;
2) add the chloroform of 800 μ l: the volume ratio of primary isoamyl alcohol is the solution of 24:1, mix 10 minutes, centrifugal 10 minutes of 10000r/min; Get supernatant liquor and add isopyknic chloroform: the volume ratio of primary isoamyl alcohol is that the solution of 24:1 is brought up again once, centrifugal 10 minutes of 10000r/min;
3) supernatant liquor is got, add the Virahol of 2 times of volumes through-20 DEG C of precoolings, slowly put upside down more than 15 times, in-20 DEG C of ice baths more than 30 minutes, then centrifugal 7 minutes of 12000r/min, incline supernatant liquor, wash 2-3 time with the ethanol of volumn concentration 70%, dehydrated alcohol is washed once, adds 200 μ l TE damping fluids, 4 DEG C of preservations after air-dry 30 minutes to drying.
6. for the CTAB-1%Tween20 Extraction buffer of the method for raising cotton seeds DNA extraction quality according to claim 1, it is characterized in that, it is: 100mmol/LTris-HCl, 20mmol/L EDTA, 2%CTAB (W/V), 2%PVP40 (W/V), 8%NaCl (W/V), 2% beta-mercaptoethanol (W/V), 1%Tween-20 (V/V).
7. for the SDS-1%Tween20 Extraction buffer of the method for raising cotton seeds DNA extraction quality according to claim 1, it is characterized in that, it is: 1%SDS (W/V), 10mmol/L EDTA, 8%NaCl (W/V), 50mmol/L Tris-HCl, 0.5% sorbyl alcohol (W/V), 1%PVP40 (W/V), 2% beta-mercaptoethanol (W/V), 1%Tween-20 (V/V).
8. the method for the raising cotton seeds DNA extraction quality described in any one of claim 1-5 is extracting the application in cotton seeds DNA.
9. CTAB-1%Tween20 Extraction buffer according to claim 6 and goods thereof are extracting the application in cotton seeds DNA.
10. SDS-1%Tween20 Extraction buffer according to claim 7 and goods thereof are extracting the application in cotton seeds DNA.
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| CN108546701A (en) * | 2018-04-28 | 2018-09-18 | 中国农业科学院棉花研究所 | A kind of method and its application of rapid extraction cotton fiber DNA |
| CN109371013A (en) * | 2018-12-02 | 2019-02-22 | 中国农业科学院植物保护研究所 | It is a kind of from insect material extract DNA extractant and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106244583A (en) * | 2016-09-23 | 2016-12-21 | 广东国盛医学科技股份有限公司 | A kind of paramagnetic particle method method for extracting nucleic acid |
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| CN108546701A (en) * | 2018-04-28 | 2018-09-18 | 中国农业科学院棉花研究所 | A kind of method and its application of rapid extraction cotton fiber DNA |
| CN109371013A (en) * | 2018-12-02 | 2019-02-22 | 中国农业科学院植物保护研究所 | It is a kind of from insect material extract DNA extractant and its application |
| CN109371013B (en) * | 2018-12-02 | 2020-07-28 | 中国农业科学院植物保护研究所 | Extracting agent for extracting DNA from insect material and application thereof |
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