CN107828783A - A kind of peanut leaf DNA highly effective extraction method - Google Patents
A kind of peanut leaf DNA highly effective extraction method Download PDFInfo
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- CN107828783A CN107828783A CN201711328951.1A CN201711328951A CN107828783A CN 107828783 A CN107828783 A CN 107828783A CN 201711328951 A CN201711328951 A CN 201711328951A CN 107828783 A CN107828783 A CN 107828783A
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- peanut leaf
- dna
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- extraction method
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The invention belongs to crop technical field, and in particular to a kind of peanut leaf DNA highly effective extraction method.The extracting method includes：Collect peanut leaf sample；Add CTAB solution, grind away in two steps into peanut leaf sample；Peanut leaf sample after grinding is put into water-bath water-bath for a period of time；Then first time extracting is carried out using chloroform/isoamyl alcohol；Reuse phenol/chloroform/isoamyl alcohol and carry out second of extracting；After extracting terminates, supernatant is taken, DNA is precipitated, is preserved after washing.The present invention can efficiently extract peanut leaf DNA under normal temperature condition, it is especially suitable for the DNA extractions of the larger material of number, less cost, shorter time can be utilized to complete a large amount of DNA batch extracting, and extraction gained DNA quality and DNA kit extraction effects are basically identical.Extracting method of the present invention is simple to operate, is reduced by more than 50 compared to kit expense.
Description
Technical field
The invention belongs to crop technical field, and in particular to a kind of peanut leaf DNA highly effective extraction method.
Background technology
The extraction of peanut tissue DNA is the first step of peanut genetic research and molecular biology experiment, DNA extraction quality and effect
The success or failure of rate direct relation subsequent experimental.Leaf tissue is that DNA extracts most-often used material.Phenolic compound is peanut leaf
One of most wide secondary metabolite of middle most species, distribution, such material easily acts on being formed surely in clasmatosis with nucleic acid
Fixed irreversible compound.At present, peanut tissue DNA extractive technique mainly has two kinds：1. use biotinylation kit, this method
It is high to extract quality, but kit is expensive, and extraction cost is higher；2. traditional CT AB methods, this method cost is relatively low, but extracts effect
Fruit is bad, can not meet subsequent experimental requirement.Both the above method is required for using liquid nitrogen during sample is ground simultaneously, leads
Cause grind away speed is slow, and extraction efficiency is low, and can be because the oxidation of the aldehydes matter in blade substantially reduce DNA quality.
The content of the invention
The present invention is directed to the deficiencies in the prior art, and it is an object of the present invention to provide a kind of extraction quality height, cost are cheap, suitable
It is suitable for the DNA highly effective extraction methods of peanut leaf.
For achieving the above object, the technical solution adopted by the present invention is：
A kind of peanut leaf DNA highly effective extraction method, it is characterised in that comprise the following steps：
(1) peanut leaf sample is taken, is put into freezen protective in centrifuge tube rapidly；
(2) CTAB solution is added in two steps into peanut leaf sample, then by the abundant grind away of peanut leaf sample；
(3) the peanut leaf sample after grinding is put into water-bath water-bath for a period of time；Then chloroform/isoamyl alcohol is used
Solution carries out first time extracting；Reuse phenol/chloroform/isoamyl alcohol and carry out second of extracting；
(4) after extracting terminates, supernatant is taken, adding NaAc and isopropanol precipitates DNA, after washing of precipitate, to precipitation
Middle RNase solution of the addition without DNase, 37 DEG C of insulation 1h, is placed at -20 DEG C and preserves.
In such scheme, step (1) the peanut leaf sample is fresh tender peanut leaf.
In such scheme, step (2) the CTAB solution is being 1%~2% using preceding needing to add volumetric concentration thereto
β-mercaptoethanol.
In such scheme, CTAB solution adds in two steps described in step (2), and the first time CTAB addition of solution is
300ul, the addition of second of CTAB solution is 600ul.
In such scheme, the mass concentration of step (2) the CTAB solution is 2%.
In such scheme, the time of grind away described in step (2) is 60s~90s.
In such scheme, the temperature of step (3) described water-bath is 65 DEG C, and the time of water-bath is 30min, and during water-bath
Every 5~10min shakes up once to peanut leaf sample.
In such scheme, the volume ratio 24 of chloroform and isoamyl alcohol in the chloroform/isoamyl alcohol:1；Phenol/the chloroform/
Phenol in isoamyl alcohol：Chloroform：The volume ratio of isoamyl alcohol is 25:24:1.
In such scheme, the NaAc of 1/10 volume and the isopropanol of isometric precooling are added in step (4) into supernatant,
Shake up, 0.5h~1h under the conditions of being placed in -20 DEG C, precipitate DNA.
In such scheme, the concentration of the NaAc is 5M/L.
Beneficial effects of the present invention：Extracting method of the present invention can efficiently extract peanut leaf under normal temperature condition
DNA, it is especially suitable for the DNA extractions of the larger material of number, can utilizing less cost, (reagent portion is traditional CT AB methods institute
Required, without other expensive reagent consumptive materials), be completed in a relatively short time DNA extractions, while can also realize to DNA sample
Batch processing, hundreds of parts of samples can be handled simultaneously, treating capacity is big, and extraction efficiency is high and DNA extractions quality is preferable.By with life
The comparison of thing kit extraction effect, the method for the invention extraction gained peanut leaf DNA quality are imitated with kit extraction
Fruit is basically identical, and the present invention effectively can extract peanut DNA under relatively simple experiment condition, is especially suitable for colony
Material makees batch extracting, simple to operate, is reduced by more than 50 compared to kit expense.
Embodiment
For a better understanding of the present invention, with reference to the embodiment content that the present invention is furture elucidated, but the present invention
Content is not limited solely to the following examples.
Embodiment 1
A kind of peanut leaf DNA highly effective extraction method, comprises the following steps：
(1) collection of peanut leaf sample：Fresh young leaflet tablet 0.2g or so is taken, is put into rapidly in 2ml centrifuge tubes and freezes
Preserve；
(2) peanut leaf is ground：Under normal temperature, (the addition before using of 300ul 2wt%CTAB solution is added in centrifuge tube
1wt% beta -mercaptoethanols), steel ball is added, (60s can fully grind blade, simultaneously using tissue sample grinding machine 60HZ grind aways 60s
Sample oxidation is avoided to greatest extent), 600ul CTAB solution (1wt% beta -mercaptoethanols are added before using) is added afterwards, such as
Need to preserve for a long time can put it into -20 DEG C of refrigerators；
(3) water-bath：Sample solution obtained by step (2) is put into warm bath 30min, every 5~10min in 65 DEG C of water sample is molten
Liquid shakes up once；
(4) chloroform/isoamyl alcohol (volume ratio 24 is used：1) solution carries out first time extracting：Add isometric (900ul) chlorine
Imitative/isoamyl alcohol (volume ratio 24：1) solution, mix, room temperature 11000r/min centrifugations 10min；
(5) phenol/chloroform/isoamyl alcohol (volume ratio 25 is used：24：1) solution carries out second of extracting：Supernatant is taken, is added
Isometric phenol/chloroform/isoamyl alcohol (volume ratio 25：24：1) solution, mix, room temperature centrifugation 12000r/min；
(6) DNA is precipitated：Supernatant is taken, the 5M/L NaAc of 1/10 volume is added, adds the isopropanol of isometric precooling, shake
It is even, -20 DEG C of refrigerator 0.5h~1h are put, precipitate DNA；Take out, 10000r/min centrifugations 10min；
(7) DNA washings, preservation：Supernatant is abandoned, precipitation is washed 2 times with 75% ethanol solution, air-dries, dissolved with 100ul TE, then
The RNase without DNase to final concentration 10ug/ul is added, 37 DEG C of insulation 1h, DNA is put in -20 DEG C afterwards and saved backup.
Heretofore described extracting method is extracted suitable for batch DNA, and the process of batch extracting is carried out to DNA and is included：(1)
The collection of each peanut leaf sample, specific method are same as above；(2) carry out batch to peanut leaf using tissue sample grinding machine to grind, have
Body method is same as above；(3) by each sample after grinding while water-bath is carried out, specific method is same as above；(4) each sample is carried out the simultaneously
Once extract, specific method is same as above；(5) each sample is carried out extracting for second simultaneously, specific method is same as above；(6) by each sample
DNA precipitations are carried out simultaneously, specific method is same as above；(7) each DNA sample washing, preservation.The method of the invention can be simultaneously to several
Hundred parts of DNA samples carry out extraction process, can be completed in a relatively short time the extraction to a large amount of DNA.
(experiment will be contrasted with DNA extraction kit extraction effect using the DNA extraction effects of the inventive method extraction
In, each material takes 2 parts of the sample of essentially identical quality, is carried respectively using the method for the invention and DNA extraction kit
Take), it is as a result as shown in table 1 below, as can be seen from Table 1：The DNA's of the 10 parts of materials extracted using the method for the invention is averaged
Concentration is 831.228ng/ul, higher than the concentration 656.533ng/ul using DNA extraction kit；Simultaneously from OD260/OD280's
Numerical value can be seen that also to be better than using DNA extraction kit using the method for the invention extraction DNA quality. (OD260/
OD280Ratio refers to absorption photometric ratio under 260nm and 280nm, for judging extraction DNA, RNA purity, the OD of pure dna260/
OD280≈1.8)。
Table 1DNA extraction effect comparison results
Obviously, above-described embodiment is only intended to clearly illustrate made example, and is not the limitation to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change or change therefore amplified
Move within still in the protection domain of the invention.
Claims (9)
1. a kind of peanut leaf DNA highly effective extraction method, it is characterised in that comprise the following steps：
(1) peanut leaf sample is taken, is put into freezen protective in centrifuge tube rapidly；
(2) CTAB solution is added in two steps into peanut leaf sample, then by the abundant grind away of peanut leaf sample；
(3) the peanut leaf sample after grinding is put into water-bath water-bath for a period of time；Then chloroform/isoamyl alcohol is used
Carry out first time extracting；Reuse phenol/chloroform/isoamyl alcohol and carry out second of extracting；
(4) after extracting terminates, supernatant is taken, adding NaAc and isopropanol precipitates DNA, will be saved backup after washing of precipitate.
2. peanut leaf DNA according to claim 1 highly effective extraction method, it is characterised in that step (1) described peanut
Leaf sample is fresh tender peanut leaf.
3. peanut leaf DNA according to claim 1 highly effective extraction method, it is characterised in that step (2) described CTAB
Solution before use, add the beta -mercaptoethanol that volumetric concentration is 1%~2% thereto.
4. peanut leaf DNA according to claim 1 highly effective extraction method, it is characterised in that step (2) described CTAB
Solution adds at twice, and the addition of first time CTAB solution is 300ul, and the addition of second of CTAB solution is 600ul.
5. peanut leaf DNA according to claim 1 highly effective extraction method, it is characterised in that the CTAB solution
Mass concentration is 2%.
6. peanut leaf DNA according to claim 1 highly effective extraction method, it is characterised in that step is ground described in (2)
The time of sample is 60s~90s.
7. peanut leaf DNA according to claim 1 highly effective extraction method, it is characterised in that step (3) described water-bath
Temperature be 65 DEG C, the time of water-bath is 30min, and every 5~10min shakes up once to peanut leaf sample during water-bath.
8. peanut leaf DNA according to claim 1 highly effective extraction method, it is characterised in that the chloroform/isoamyl alcohol
The imitative volume ratio 24 with isoamyl alcohol of Chlorine in Solution:1；Phenol in the phenol/chloroform/isoamyl alcohol：Chloroform：The volume ratio of isoamyl alcohol
For 25:24:1.
9. peanut leaf DNA according to claim 1 highly effective extraction method, it is characterised in that to supernatant in step (4)
The NaAc of 1/10 volume and the isopropanol of isometric precooling are added in liquid, is shaken up, 0.5h~1h under the conditions of being placed in -20 DEG C, precipitation
DNA。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410865A (en) * | 2018-05-31 | 2018-08-17 | 山东省花生研究所 | One method for cultivating peanut DNA rapid extractions |
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Application publication date: 20180323 |