CN107699561A - A kind of modified CTAB method extracts cotton genomic dna - Google Patents
A kind of modified CTAB method extracts cotton genomic dna Download PDFInfo
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Abstract
The invention discloses a kind of modified CTAB method to extract cotton genomic dna, comprises the following steps：（1）Liquid nitrogen smashes, and extract solution I that must be containing DNA is shaken in addition CTAB 65 DEG C of water-baths of buffer solution；（2）37 DEG C of water-baths are carried out after RNase A are added after cooling；（3）Add isometric solution A mixing and centrifuge to obtain supernatant I；（4）Continuously add isometric solution A mixing and centrifuge to obtain supernatant II；（5）NaAc is added, horizontal shake of absolute ethyl alcohol is then added and forms flocculent deposit；（6）Centrifugation, supernatant is abandoned, washing is heavy to discard absolute ethyl alcohol, dry to precipitate DNA；（7）Dissolving precipitation DNA, is stored in standby in 20 DEG C of refrigerators.The present invention has simplified the step program of CTAB methods extraction cotton leaf genomic DNA, substantially reduces experimental period, also reduces DNA extraction costs, improves RNA eradicating efficacy, is adapted to the high-volume extraction of cotton leaf genomic DNA.
Description
Technical field
The present invention relates to DNA extractive techniques field, and in particular to a kind of modified CTAB method extracts cotton genomic dna.
Background technology
In recent years, carried out in a deep going way with Crop Quantitative Traits gene loci Position Research, molecular marking technique is increasingly
Crop marker-assisted breeding is widely applied to by scientific research and breeding units.In crop marker-assisted breeding program, high flux,
Quickly, the DNA of low cost extraction crop individual plant is always scientific research and the dreamboat of breeder.Aid in selecting in cotton molecular labeling
Select in work, usually extract DNA sample from cotton plant blade, unlike the DNA propositions of other plant, in cotton leaf
In addition to rich in albumen, also hold containing the secondary metabolites such as more gossypol, polysaccharide, tannin, these metabolins in cell rupture
It is oxidizable, irreversible reaction occurs with protein, nucleic acid etc. after oxidation, forms brown gum compound, influences follow-up experiment
The step of working, therefore causing cotton leaf DNA to extract is increasingly complex compared with other crops.
At present, conventional cotton leaf DNA extracting methods are CTAB methods.This method includes field sampling, tissue is broken
Broken, Extraction buffer pretreatment, lysis buffer cell lysis, remove the impurity such as isolating protein and RNA, precipitation DNA, rinsing DNA
With dissolving etc. link.But traditional CTAB methods, when RNA is purged in cotton gene group, it is necessary to which DNA is dissolved after
Extract again one time, this makes extraction step become very very complicated, and time-consuming and DNA yield is low.
Also there is kit extraction cotton leaf DNA of the laboratory using commercialization, the kit of commercialization usually exists
Add RNaseA in SolutionI, cross before post and just to cut RNA into pieces sections, and RNA generally less than normal, mistakes of comparing genomic DNA
It can not be suspended to during post on post, therefore be able to remove RNA.Although the kit RNA removal effects of commercialization are good, the consuming time is short,
But cost is high, large batch of material process is not appropriate for.
In recent years, gene order-checking research was paid attention to further both at home and abroad, each state all increases research dynamics of investment.But base
Because group sequencing is high to the quality requirement of genomic DNA, RNA presence has had a strong impact on the quality of gene order-checking, and often
Need to handle substantial amounts of sample in the short time, it is therefore desirable to a kind of to simplify the Plant Genome extracting method for removing RNA.
The content of the invention
It is an object of the invention to provide a kind of modified CTAB method to extract cotton genomic dna, to solve existing DNA
The side's of extraction technical step is cumbersome, and RNA removes the technical problem that time-consuming.
To achieve the above object, the technical scheme is that a kind of modified CTAB method extracts cotton genomic dna, including
Following steps：
（1）Draw materials, smashed with liquid nitrogen, move into centrifuge tube, fully shaken up after adding CTAB buffer solutions, in 65 DEG C of water-baths
Middle reaction 40 ~ 60 minutes, gently shakes every 10min, obtains the extract solution I containing DNA；
（2）The extract solution I centrifuge tubes for filling DNA are placed after cooling in ice, add the laggard water-filling baths of RNase A；
（3）Isometric solution A vibration is added into reacted DNA extract solution I and mixes 3min, 10000r/min centrifugations
10min obtains supernatant I；The solution A is by chloroform：Isoamyl alcohol is according to 24:1 volume ratio mixes；
（4）Isometric solution A is added in upward clear liquid I, vibration mixes 3min, and 10000r/min centrifugations 10min obtains supernatant
II；
（5）The 3M NaAc of 0.1 times of volume are added in upward clear liquid I I, are then slowly added into the nothing of -20 DEG C of precoolings of 2 times of volumes
Water-ethanol, level shake centrifuge tube 5min and form flocculent deposit；
（6）5min is centrifuged at 10000 r/min, 4 DEG C, abandons supernatant, cleans precipitation twice with 70% ethanol, absolute ethyl alcohol is clear
Wash precipitation once, discard absolute ethyl alcohol, it is dry to precipitate DNA；
（7）Dissolving precipitation DNA, is stored in standby in -20 DEG C of refrigerators.
Preferably, step（1）In, the blade of the material selection upland cotton.
Preferably, step（1）In, the material is with CTAB buffer solutions according to m:V is 1 ~ 2:10 are mixed.
Preferably, step（1）In, contain in the CTAB buffer solutions：1.4 M NaCl, 0.02 M EDTA, 0.1 M
Tris-HCl, 2% cetyl trimethylammonium bromide, 2% PVP-K30,0.1% DIECA, 0.2% beta -mercaptoethanol, solvent are
Deionized water；Wherein, the beta -mercaptoethanol used time now adds.
Preferably, step（2）In, the extract solution I centrifuge tubes for filling DNA are placed after cooling in ice, according to volume basis
Than in the extract solution I to DNA add 0.25% ~ 1% RNase A, then under 27 ~ 37 DEG C of water bath conditions reaction 7.5 ~
60min。
Preferably, step（2）In, the extract solution I centrifuge tubes for filling DNA are placed after cooling in ice, according to volume basis
Than the RNase A that 0.5% is added in the extract solution I to DNA, then 7.5min is reacted under 37 DEG C of water bath conditions.
Preferably, step（2）In, the extract solution I centrifuge tubes for filling DNA are placed after cooling in ice, according to volume basis
Than the RNase A that 0.25% is added in the extract solution I to DNA, then 7.5min is reacted under 37 DEG C of water bath conditions.
Preferably, step（7）In, with 100 μ l sterilizing ultra-pure water dissolving precipitations DNA.
Preferably, step（7）In, precipitate DNA with 50 ~ 100 μ l TE buffer solutions；Contain 1M in the TE buffer solutions
TrisCl, 0.5M EDTA, solvent are deionized water, and the molten pH of the TE buffer solutions is 8.0.
The invention provides a kind of modified CTAB method to extract cotton genomic dna, and it is using CTAB solution in different temperatures
The difference of lower cracking ability plays RNase A effect, removes RNA present in genomic DNA, simplifies Plant Genome RNA
Reset procedure.The inventive method has the following advantages that：
（1）Compared to first completed in existing extracting method DNA extraction after again individually carry out RNA removings, the present invention around and
Row, reduce operating procedure and program, saved cost, substantially reduce DNA extraction time, accelerate experiment progress.
（2）The present invention shifts to an earlier date RNase A digestion, and the DNA without being completed to extraction is digested and extracted again, and is subtracted
Lack the extracting number of extract solution and the washing times of DNA precipitations, ensure that DNA yield.
（3）The RNA eradicating efficacies of the present invention are good, and the DNA mass of acquisition is high and stably, is advantageous to follow-up experimental study.
（4）The present invention modified CTAB method not only suitable for cotton leaf genome RNA elimination, it can also be used to cotton
Other crops such as flower, corn, wheat, soybean, rice, tobacco.
The present invention has simplified the step program of CTAB methods extraction cotton leaf genomic DNA, substantially reduces experimental period,
DNA extraction costs are also reduced, improve RNA eradicating efficacy, are adapted to the high-volume extraction of cotton leaf genomic DNA.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis figure of the embodiment of the present invention 2,
Swimming lane 1 is that Tiangeng DP320 kits extract upland cotton leaves genomic DNA, RNase A bath temperature in swimming lane 2 ~ 6
Respectively 22 DEG C, 27 DEG C, 32 DEG C, 37 DEG C, 65 DEG C；
Fig. 2 is the agarose gel electrophoresis figure of the embodiment of the present invention 3,
Swimming lane 1 is that Tiangeng DP320 kits extract upland cotton leaves genomic DNA, RNase A digestion time in swimming lane 2 ~ 6
Respectively 2h, 1h, 0.5h, 15min, 7.5min；
Fig. 3 is the agarose gel electrophoresis figure of the embodiment of the present invention 4,
RNase A usage amount is respectively 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125% in swimming lane 1 ~ 6（v/v）.
Fig. 4 is the agarose gel electrophoresis figure of the embodiment of the present invention 5,
Swimming lane 1 ~ 3 is the upland cotton leaves genomic DNA obtained by the method for embodiment 4, and swimming lane 4 ~ 6 is to pass through the side of embodiment 5
The upland cotton leaves genomic DNA that method obtains；
Fig. 5 is the agarose gel electrophoresis figure of embodiments of the invention 6 ~ 10,
Swimming lane 1 ~ 5 is respectively corn, wheat, soybean, rice, the genomic DNA of tobacco.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
The extracting genome DNA of the upland cotton blade of embodiment 1
A kind of modified CTAB method extracts cotton genomic dna, comprises the following steps：
（1）Using the blade of upland cotton as experiment material, take 100 ~ 200 mg samples to be fitted into 2.0mL centrifuge tubes, ground with liquid nitrogen
Into powder, fully shake up after then adding 1000 μ L CTAB buffer solutions, reacted 40 ~ 60 minutes in 65 DEG C of water-baths, every
10min gently shakes, and obtains the extract solution I containing DNA.The powder is with CTAB buffer solutions according to m:V is 1 ~ 2:10 are carried out
Mixing.
Contain in the CTAB buffer solutions：1.4 M NaCl（Sodium chloride）, 0.02 M EDTA（pH8.0）, 0.1 M
Tris-HCl（pH7.5）, 2%（w/v）Cetyl trimethylammonium bromide（CTAB）, 2%（w/v）PVP-K30,0.1%（w/v）
DIECA, 0.2%（v/v）Beta -mercaptoethanol, solvent are deionized water, wherein, beta -mercaptoethanol is current now to be added.
（2）The extract solution I centrifuge tubes for filling DNA are placed after cooling in ice, add the laggard water-filling baths of RNase A.
（3）Isometric solution A vibration is added into reacted DNA extract solution I and mixes 3min, 10000r/min from
Heart 10min obtains supernatant I；The solution A is by chloroform：Isoamyl alcohol is according to 24:1 volume ratio mixes.
（4）Add isometric solution A in upward clear liquid I, vibration mixes 3min, 10000r/min centrifugations 10min must on
Clear liquid I I.
（5）0.1 times of volume is added in upward clear liquid I I（Account for supernatant II 1/10 volume）3M NaAc（PH5.2）,
It is then slowly added into the absolute ethyl alcohol of -20 DEG C of precoolings of 2 times of volumes（I.e. absolute ethyl alcohol is supernatant II and NaAc volume sums
2 times）, horizontally rotate centrifuge tube 5min and form flocculent deposit.
（6）5min is centrifuged at 10000 r/min, 4 DEG C, abandons supernatant, precipitation is washed 2 times with 70% ethanol, then with anhydrous
Ethanol washing precipitation 1 time, discards absolute ethyl alcohol, DNA must be precipitated by drying or drying in vacuum drier.
（7）With 100 μ l sterilizing ultra-pure water dissolving precipitation DNA, it is stored in standby in -20 DEG C of refrigerators.
In the step, it is also possible to which 50 ~ 100 μ l TE buffer solutions precipitate DNA.Contain 1M in the TE buffer solutions
TrisCl, 0.5M EDTA, solvent are deionized water, and the molten pH of the TE buffer solutions is 8.0.
The determination of the RNase A operative temperatures of embodiment 2
Prepare more parts of upland cotton blade materials, the genomic DNA of upland cotton blade is extracted using the method for embodiment 1.Wherein, exist
Step（2）In, the extract solution I centrifuge tubes for filling DNA are placed after cooling in ice, be separately added into 1%（v/v）RNase A（Purchase
From precious biotech firm, specification is 10mg/ml）, then disappear respectively under 22 DEG C, 27 DEG C, 32 DEG C, 37 DEG C, 65 DEG C of water bath condition
Change 2h.
The Ago-Gel of configuration 0.8%, electrophoresis is carried out with 1 × TAE electrophoretic buffers to the genome DNA of extraction
Detection, 1 × TAE electrophoretic buffers composition are as follows：0.04mol/LTris- acetic acid, 0.001mol/L EDTA.With reagent
Box extracts upland cotton leaves genomic DNA（Tiangeng DP320）As control, the upland cotton leaf obtained with said temperature gradient experiment
Piece genomic DNA enters row agarose gel electrophoresis together.Draw the μ L of genomic DNA 5, and 1ul 6 × sample-loading buffer (treasured
Biotech firm produces) mixing, after being well mixed, according to 15v/cm voltage stabilizings electrophoresis 30 minutes, electrophoresis terminate after with gel imaging system
Unite to observe, as a result see Fig. 1.As shown in figure 1, the genomic DNA band obtained by the modified CTAB method of the present invention is clearly complete
It is whole, the effect of kit extraction can be reached, also, at 27 ~ 37 DEG C, RNase action effect is good, especially at 37 DEG C,
RNA removal effects are best, can't see dying RNA bands completely.
The determination of the RNase A action times of embodiment 3
Prepare more parts of upland cotton blade materials, the genomic DNA of upland cotton blade is extracted using the method for embodiment 1.Wherein, exist
Step（2）In, the extract solution I centrifuge tubes for filling DNA are placed after cooling in ice, be separately added into 1%（v/v）RNase A, so
Digest 2h, 1h, 0.5h, 15min, 7.5min under 37 DEG C of water bath condition respectively afterwards.
Upland cotton leaves genomic DNA is extracted with kit（Tiangeng DP320）As control, disappear with above-mentioned RNase differences
Change the upland cotton leaves genomic DNA obtained under time effect and enter row agarose gel electrophoresis together, as a result see Fig. 2.Such as Fig. 2 institutes
Show, 1%（v/v）The RNase A of concentration water-bath 7.5min under the conditions of 37 DEG C are enough the RNA in fully digestion genome.
The determination of the RNase A activities of embodiment 4
Prepare more parts of upland cotton blade materials, the genomic DNA of upland cotton blade is extracted using the method for embodiment 1.Wherein, exist
Step（2）In, the extract solution I centrifuge tubes for filling DNA are placed after cooling in ice, be separately added into 1%（v/v）、0.5%（v/v）、
0.25%（v/v）、0.125%（v/v）、0.0625%（v/v）、0.03125%（v/v）RNase A, then respectively in 37 DEG C of water
7.5min is digested under the conditions of bath.
The upland cotton leaves genomic DNA obtained under above-mentioned different RNase A additions is subjected to Ago-Gel together
Electrophoresis, as a result see Fig. 3.As shown in figure 3,0.25%（v/v）The following concentration of RNase A can not digest the RNA in genome completely.
Therefore draw, the optimal conditions of RNase A digestion：0.25% is added in DNA extract solution I（v/v）RNase A are at 37 DEG C
Lower water-bath 7.5min.
As can be seen here, modified CTAB method not only reduces reactions steps, and saves RNase A usage amount, shortens
Digestion time, so as to reduce sample process cost, improves sample process efficiency.Present invention supposition, the CTAB methods of improvement
In RNA digestion processes, because lysate crushes cell membrane, the endogenous RNase A of plant cell are discharged therewith, with
The RNase A of external source addition are acted synergistically, and so as to reduce exogenous RNase A usage amount, and shorten RNA digestion
Time.
The comparative example of embodiment 5
A kind of modified CTAB method extracts cotton genomic dna, comprises the following steps：
（1）Draw materials, smashed with liquid nitrogen.
（2）The powder of grinding number is moved into centrifuge tube, fully shaken up after adding CTAB buffer solutions, it is anti-in 65 DEG C of water-baths
Answer 40 ~ 60 minutes, gently shaken every 10min, obtain the extract solution I containing DNA.
（3）Isometric solution A vibration is added into reacted DNA extract solution I and mixes 3min, 10000r/min from
Heart 10min obtains supernatant I；The solution A is by chloroform：Isoamyl alcohol is according to 24:1 volume ratio mixes.
（4）Isometric solution A is added in upward clear liquid I, vibration mixes 3min, 10000r/min centrifugation 10min, obtained
Clear liquid I I.
（5）The isopropanol of -20 DEG C of precoolings of 0.6 times of volume is slowly added in upward clear liquid I I, level is shaken centrifuge tube, sunk
Shallow lake 30min.
（6）5min is centrifuged at 10000 r/min, 4 DEG C, abandons supernatant, cleans precipitation twice with 70% ethanol, absolute ethyl alcohol
Cleaning precipitation once, discards absolute ethyl alcohol, dry to precipitate DNA；
（7）Dissolving precipitation DNA, forms DNA lysates I.
（8）1% is added into DNA lysates I（v/v）RNase A, 37 DEG C of water-bath 2h.
（9）Isometric solution A is added to DNA lysates I, vibration mixes 3min, 10000r/min centrifugation 10min, obtained
Supernatant III.
（10）The 3M NaAc of 0.1 times of volume are added in upward clear liquid I II, be then slowly added into 2 times of volumes -20 DEG C are pre-
Cold absolute ethyl alcohol, level shake centrifuge tube 5min and form flocculent deposit.
（11）5min is centrifuged at 10000 r/min, 4 DEG C, abandons supernatant, cleans precipitation twice with 70% ethanol, anhydrous second
Alcohol cleaning precipitation once, discards absolute ethyl alcohol, dry to precipitate DNA.
（12）Dissolving precipitation DNA, is stored in standby in -20 DEG C of refrigerators.
3 are taken respectively by the present embodiment（Traditional CTAB methods）The upland cotton leaves genomic DNA of preparation and by above-mentioned
Embodiment 4（RNA digestion conditions are：0.25% is added in DNA extract solution I（v/v）RNase A water-baths at 37 DEG C
7.5min）The upland cotton leaves genomic DNA of preparation as a control group, enters row agarose gel electrophoresis, as a result sees Fig. 4.Such as Fig. 4
Understand, the modified CTAB method of traditional CTAB methods and the present invention, which can remove the RNA in cotton leaf genomic DNA, to be done
Only, still, the band brightness for the genomic DNA that the CTAB methods of improvement obtain is not weaker than the genomic DNA of traditional CT AB methods acquisition
Band.
Use ultramicrospectrophotometer (model：Eppendorf BioPhotometer Plus) present invention is implemented
The cotton leaf DNA and embodiment 5 of the modified CTAB method extraction of example 4（Traditional CTAB methods）The cotton leaf DNA of extraction
Concentration mensuration has been carried out, and DNA concentration, time-consuming and cost comparison have been carried out to two kinds of extraction DNA method, has as a result been seen
Table 1.
The comparison of the upland cotton leaves genomic DNA sample concentration of the two methods of table 1. extraction
| Extracting method | Concentration（ng/ul） | It is time-consuming（Hour/experiment） |
| Improved method | 300±20 | 2 |
| Traditional Method | 200±20 | 5 |
From table 1 it follows that using modified CTAB method of the present invention extraction DNA samples content with it is existing traditional
The content of the cotton DNA of CTAB methods extraction compares the raising for having 50%；And relative to traditional CTAB methods, every group of sample
Processing time saved as many as 3 hours；And due to reducing an extraction steps and 3 washing of precipitate numbers,
RNaseA dosage and digestion time is also few, reduces the usage amount of kit enzyme, so as to save extraction cost.
The present invention speculates, the extracting number of solution A and the washing time of DNA precipitations are reduced due to the CTAB methods after improvement
Number, reduces DNA loss, so as to improve DNA yield.And from the point of view of time comparative result used in sample extraction, this
The time-consuming of invention is far below traditional CTAB methods, therefore within the equal time, utilizes the sample of the method processing of the present invention
Measure far more than traditional CTAB methods.
The extraction of the maize leaf genomic DNA of embodiment 6
Maize leaf genomic DNA is extracted using the method for the present invention, then enters row agarose gel electrophoresis, as a result sees
Fig. 5.
The extraction of the wheat leaf blade genomic DNA of embodiment 7
Wheat leaf blade genomic DNA is extracted using the method for the present invention, then enters row agarose gel electrophoresis, as a result sees
Fig. 5.
The extraction of the soybean leaves genomic DNA of embodiment 8
Soybean leaves genomic DNA is extracted using the method for the present invention, then enters row agarose gel electrophoresis, as a result sees
Fig. 5.
The extraction of the rice leaf genomic DNA of embodiment 9
Rice leaf genomic DNA is extracted using the method for the present invention, then enters row agarose gel electrophoresis, as a result sees
Fig. 5.
The extraction of the tobacco leaf genomic DNA of embodiment 10
Tobacco leaf genomic DNA is extracted using the method for the present invention, then enters row agarose gel electrophoresis, as a result sees
Fig. 5.
As shown in Figure 5, by the present invention modified CTAB method extract corn, wheat, soybean, rice, tobacco genome
DNA, electrophoresis result prove that its genomic DNA band complete display obtained, RNA is removed also very clean, and DNA obtains quality
It is high.Hence it is demonstrated that the present invention is not only applied to the extraction of the upland cotton genomic DNA rich in polysaccharide polyphenol, in other crops
Effect same is good in extracting genome DNA.Also, the present invention, compared to existing CTAB methods, step simply takes short, extraction
Efficiently, the labor intensity of scientific research personnel can be substantially reduced.
Although above with general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, 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, belong to the scope of protection of present invention.
Claims (9)
1. a kind of modified CTAB method extracts cotton genomic dna, it is characterised in that comprises the following steps：
（1）Draw materials, smashed with liquid nitrogen, move into centrifuge tube, fully shaken up after adding CTAB buffer solutions, in 65 DEG C of water-baths
Middle reaction 40 ~ 60 minutes, gently shakes every 10min, obtains the extract solution I containing DNA；
（2）The extract solution I centrifuge tubes for filling DNA are placed after cooling in ice, add the laggard water-filling baths of RNase A；
（3）Isometric solution A vibration is added into reacted DNA extract solution I and mixes 3min, 10000r/min centrifugations
10min obtains supernatant I；The solution A is by chloroform：Isoamyl alcohol is according to 24:1 volume ratio mixes；
（4）Isometric solution A is added in upward clear liquid I, vibration mixes 3min, and 10000r/min centrifugations 10min obtains supernatant
II；
（5）The 3M NaAc of 0.1 times of volume are added in upward clear liquid I I, are then slowly added into the nothing of -20 DEG C of precoolings of 2 times of volumes
Water-ethanol, level shake centrifuge tube 5min and form flocculent deposit；
（6）5min is centrifuged at 10000 r/min, 4 DEG C, abandons supernatant, cleans precipitation twice with 70% ethanol, absolute ethyl alcohol is clear
Wash precipitation once, discard absolute ethyl alcohol, it is dry to precipitate DNA；
（7）Dissolving precipitation DNA, is stored in standby in -20 DEG C of refrigerators.
2. modified CTAB method according to claim 1 extracts cotton genomic dna, it is characterised in that：Step（1）In, institute
State the blade of material selection upland cotton.
3. modified CTAB method according to claim 2 extracts cotton genomic dna, it is characterised in that：Step（1）In, institute
Material is stated with CTAB buffer solutions according to m:V is 1 ~ 2:10 are mixed.
4. modified CTAB method according to claim 3 extracts cotton genomic dna, it is characterised in that：Step（1）In, institute
State and contain in CTAB buffer solutions：1.4 M NaCl, 0.02 M EDTA, 0.1 M Tris-HCl, 2% cetyl trimethyl bromine
Change ammonium, 2% PVP-K30,0.1% DIECA, 0.2% beta -mercaptoethanol, solvent is deionized water；Wherein, beta -mercaptoethanol is used
Shi Xianjia.
5. modified CTAB method according to claim 3 extracts cotton genomic dna, it is characterised in that：Step（2）In, will
Fill after cooling in DNA extract solution I centrifuge tubes placement ice, 0.25% is added into DNA extract solution I according to percent by volume
~ 1% RNase A, 7.5 ~ 60min is then reacted under 27 ~ 37 DEG C of water bath conditions.
6. modified CTAB method according to claim 3 extracts cotton genomic dna, it is characterised in that：Step（2）In, will
Fill after cooling in DNA extract solution I centrifuge tubes placement ice, 0.5% is added into DNA extract solution I according to percent by volume
RNase A, then react 7.5min under 37 DEG C of water bath conditions.
7. modified CTAB method according to claim 3 extracts cotton genomic dna, it is characterised in that：Step（2）In, will
Fill after cooling in DNA extract solution I centrifuge tubes placement ice, 0.25% is added into DNA extract solution I according to percent by volume
RNase A, then react 7.5min under 37 DEG C of water bath conditions.
8. the modified CTAB method extraction cotton genomic dna according to any one of claim 5 ~ 7, it is characterised in that：Step
（7）In, with 100 μ l sterilizing ultra-pure water dissolving precipitations DNA.
9. the modified CTAB method extraction cotton genomic dna according to any one of claim 5 ~ 7, it is characterised in that：Step
（7）In, precipitate DNA with 50 ~ 100 μ l TE buffer solutions；It is molten containing 1M TrisCl, 0.5M EDTA in the TE buffer solutions
Agent is deionized water, and the molten pH of the TE buffer solutions is 8.0.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114426967A (en) * | 2020-10-29 | 2022-05-03 | 西南大学 | Non-toxic plant universal high-purity genome DNA rapid extraction method and kit |
| CN114507661A (en) * | 2022-03-14 | 2022-05-17 | 吉林农业大学 | Rapid extraction method of genomic DNA of acer negundo |
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| CN114507661A (en) * | 2022-03-14 | 2022-05-17 | 吉林农业大学 | Rapid extraction method of genomic DNA of acer negundo |
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