CN104894104B - A kind of method for the genomic DNA for being suitable for extraction banana different tissues - Google Patents
A kind of method for the genomic DNA for being suitable for extraction banana different tissues Download PDFInfo
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Abstract
The method of the genomic DNA for being suitable for extraction banana different tissues of the present invention includes：Banana Tissue sample is taken, liquid nitrogen grinding is added into powder, goes in centrifuge tube rapidly；Then, Extraction buffer, 10%SDS and benzyl chloride, water-bath after fully mixing are added；Then, sodium acetate ice bath is added, is then centrifuged for；The supernatant learnt from else's experience obtained by previous step adds the isopropanol of precooling, is centrifuged after mixing, abandon supernatant to a clean centrifuge tube；The precipitation that previous step of learning from else's experience obtains is washed with 75% ethanol, and room temperature volatilization ethanol, then adds TE buffer, then is digested with suitable RNase A, takes out and is saved backup after 20 DEG C.The above method limits from drawing materials, the genomic DNA of energy rapid extraction high quality, and the purity height and amount of the genomic DNA extracted from unit Banana Tissue sample are big from banana different tissues organ.
Description
Technical field
The invention belongs to biochemical field, and in particular to a kind of genomic DNA for being suitable for extraction banana different tissues
Method.
Background technology
DNA molecular marker technology is widely used in plant germplasm resource research.Can be big by molecular mark
It is big to shorten breeding cycle, influenced from factors such as developmental stage, histoorgan and environmental conditions.Efficiently, high quality is quickly prepared
Genomic DNA, and ensure the purity and integrality of DNA, be to carry out molecular labeling and the basis of other molecular biology tests.
From plant tissue extract genomic DNA common method have CTAB (cetyltriethylammonium bromide) methods and
SDS (dodecyl sodium sulfate) method, and the improved method based on both approaches.These methods are suitable for banana tender tissue
Extracting genome DNA, the DNA purity of extraction disclosure satisfy that the needs of general molecular biology, but for rich in polysaccharide, polyphenol
Needs are repeatedly extracted by substep centrifugation, or addition potassium acetate and chloroform/isoamyl alcohol for banana, not only extraction effect
It cannot be guaranteed that and operating procedure it is complicated, time-consuming, easy cross contamination.Corresponded in existing experimental implementation using a kind of tissue
A kind of mode of method extracts the genomic DNA of plant different tissues, since every kind of method medicine to be configured is different, operation step
It is rapid different, therefore experimental procedure can be increased, increase workload.
In view of the foregoing, it is expected to obtain a kind of gene of equal energy rapid extraction high quality in different tissues organ from banana
The method of group DNA, i.e. only a kind of method of application just can extract the genomic DNA in banana different tissues organ.
The content of the invention
Present invention seek to address that the above problem.
It is an object of the present invention to provide a kind of method for the genomic DNA for being suitable for extraction banana different tissues.
The genomic DNA method for being suitable for extraction banana different tissues of the present invention includes the following steps：Step 1：Take perfume (or spice)
Any of several broadleaf plants tissue sample, adds grind into powder after liquid nitrogen, goes in centrifuge tube；Step 2：In the banana group of the step 1
Extraction buffer, 10%SDS and benzyl chloride, water-bath after fully mixing are added in tissue samples；Step 3：Obtained through the step 2
To mixed liquor in add sodium acetate ice bath, be then centrifuged for separating；Step 4：The supernatant learnt from else's experience obtained by the step 3 is to one dry
Net centrifuge tube, adds the isopropanol of precooling, is centrifuged after mixing, abandon supernatant；Step 5：Learn from else's experience what the step 4 obtained
Precipitation wash with 75% ethanol, and room temperature is volatilized ethanol, then addition TE buffer or sterile water, then with suitable RNase A
Digestion, takes out and is saved backup after -20 DEG C.
Further, the dosage of Banana Tissue sample described in the step 1 is 0.5g；Carried described in the step 2
The dosage for taking buffer solution is 1mL, the dosage of the 10%SDS is 150 μ L, the dosage of the benzyl chloride is the μ L of 200 μ L~800；
The content that the sodium acetate is added in the step 3 is 450 μ L；The dosage of the isopropanol in the step 4 is
0.1mL。
Further, the benzyl chloride is 600 μ L.
Further, the Extraction buffer includes the 100mmolL of pH 9.0-1Tris-HCl, pH's 8.0
40mmol·L-1EDTA。
Further, bath temperature is 65 DEG C in the step 2, water bath time 45min.
Further, the time of mixed liquor described in sodium acetate ice bath described in the step 3 is 15min, and set from
Heart parameter centrifuges 8min for 13000 × g.
Further, the parameter of noncentricity set in the step 4 centrifuges 10min as 13500 × g.
Further, 75% ethanol described in the step 5 washs the number of the precipitation as twice.
The beneficial effects of the present invention are：The genomic DNA method for being suitable for extraction banana different tissues of the present invention is not
Limited by materials, from banana different tissues organ can rapid extraction high quality genomic DNA, and from unit banana group
The purity height and high income of the genomic DNA extracted in tissue samples.
Brief description of the drawings
Fig. 1-1 shows that CTAB conventional methods according to prior art and CTAB improved methods 1 extract banana and respectively organize genomic DNA
Agarose gel electrophoresis figure；
Fig. 1-2 shows that CTAB improved methods 3 according to prior art and CTAB improved methods 2 extract banana and respectively organize genome
The agarose gel electrophoresis figure of DNA；
Fig. 1-3 shows that SDS methods according to prior art and SDS associated proteins enzyme K methods extraction banana respectively organize genomic DNA
Agarose gel electrophoresis figure；
Fig. 1-4 shows that improveing urea method and Benzyl chloride method according to the present invention extraction banana according to prior art respectively organizes
The agarose gel electrophoresis figure of genomic DNA；
Fig. 2 shows that various concentrations benzyl chloride extraction banana respectively organizes genomic DNA in Benzyl chloride method according to the present invention
Agarose gel electrophoresis result；
Fig. 3 shows the ITS amplified production agarose gel electrophoresis figures of Benzyl chloride method DNA according to the present invention.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.It should be noted that the skill described in following embodiments
The combination of art feature or technical characteristic is not construed as isolated, they can be mutually combined so as to reaching more preferable
Technique effect.
The comparative experiments of banana genome DNA extraction method
In banana genome DNA extraction method comparative experiments, the leaf of banana, false stem, root and fruit sample pick up from state
Interior banana main breed ' Brazilian any of several broadleaf plants ' (Musa Cavendish AAA).In addition, respectively to pericarp and pulp portion after the accelerating ripening of fruit
Position is sampled.After all samples collection it is rapid with 95% alcohol washes, be respectively placed in -80 DEG C of ultra low temperature freezers preserve it is standby
With.
The above-mentioned each sample of 0.5g is taken, adds grind into powder after appropriate liquid nitrogen, is gone to rapidly in 2mL centrifuge tubes, Ran Houfen
Genomic DNA is not extracted as follows.
(1) Benzyl chloride method (i.e. method of the invention)
Step is as follows：
Embodiment 1：
1. add 1mL Extraction buffers (100mmolL-1Tris-HCl pH 9.0,40mmolL-1EDTA pH
8.0) 65 DEG C of water-bath 45min after, and 200 μ L of 150 μ L 10%SDS and benzyl chloride, fully mixing, during which shake up every 10min
Once；
2. adding 450 μ L sodium acetate ice bath 15min, centrifuged under conditions of the parameter of noncentricity set is 13000 × g
8min；
3. supernatant is taken to add the isopropanol of 0.1mL-20 DEG C of precooling to a new centrifuge tube, set after mixing of turning upside down
Parameter of noncentricity be 13500 × g under conditions of centrifuge 10min, abandon supernatant；
4. taking precipitation to be washed 2 times with 75% ethanol, room temperature is volatilized ethanol, with TE buffer or sterile water dissolving DNA,
(37 DEG C of water-bath 1h) is digested with suitable RNase A again, takes out and is saved backup after -20 DEG C.
Embodiment 2：
1. add 1mL Extraction buffers (100mmolL-1Tris-HCl pH 9.0,40mmolL-1EDTA pH
8.0) 65 DEG C of water-bath 45min after, and 600 μ L of 150 μ L 10%SDS and benzyl chloride, fully mixing, during which shake up every 15min
Once；
2. adding 450 μ L sodium acetate ice bath 15min, centrifuged under conditions of the parameter of noncentricity set is 13000 × g
8min；
3. supernatant is taken to add the isopropanol of 0.1mL-20 DEG C of precooling to a new centrifuge tube, set after mixing of turning upside down
Parameter of noncentricity be 13500 × g under conditions of centrifuge 10min, abandon supernatant；
4. taking precipitation to be washed 2 times with 75% ethanol, room temperature is volatilized ethanol, with TE buffer or sterile water dissolving DNA,
(37 DEG C of water-bath 2h) is digested with suitable RNase A again, takes out and is saved backup after -20 DEG C.
Embodiment 3：
1. add 1mL Extraction buffers (100mmolL-1Tris-HCl pH 9.0,40mmolL-1EDTA pH
8.0) 65 DEG C of water-bath 45min after, and 800 μ L of 150 μ L 10%SDS and benzyl chloride, fully mixing, during which every 10-15min
Shake up once；
2. adding 450 μ L sodium acetate ice bath 15min, centrifuged under conditions of the parameter of noncentricity set is 13000 × g
8min；
3. supernatant is taken to add the isopropanol of 0.1mL-20 DEG C of precooling to a new centrifuge tube, set after mixing of turning upside down
Parameter of noncentricity be 13500 × g under conditions of centrifuge 10min, abandon supernatant；
4. taking precipitation to be washed 2 times with 75% ethanol, room temperature is volatilized ethanol, with TE buffer or sterile water dissolving DNA,
(37 DEG C of water-bath 1-2h) is digested with suitable RNase A again, takes out and is saved backup after -20 DEG C.
Embodiment 4：
1. add 1mL Extraction buffers (100mmolL-1Tris-HCl pH 9.0,40mmolL-1EDTA pH
8.0) 65 DEG C of water-bath 45min after, and 400 μ L of 150 μ L 10%SDS and benzyl chloride, fully mixing, during which every 10-15min
Shake up once；
2. adding 450 μ L sodium acetate ice bath 15min, centrifuged under conditions of the parameter of noncentricity set is 13000 × g
8min；
3. supernatant is taken to add the isopropanol of -20 DEG C of precoolings to a new centrifuge tube, turn upside down after mixing in the centrifugation of setting
Parameter centrifuges 10min under conditions of being 13500 × g, abandons supernatant；
4. taking precipitation to be washed 2 times with 75% ethanol, room temperature is volatilized ethanol, with TE buffer or sterile water dissolving DNA,
(37 DEG C of water-bath 1-2h) is digested with suitable RNase A again, takes out and is saved backup after -20 DEG C.
(2) CTAB conventional methods
Step is as follows：
1. add the CTAB Extraction buffers (100mmolL of 65 DEG C of preheatings of 1mL-1Tris-HCl pH 8.0、
20mmol·L-1EDTA pH 8.0、1.4mol·L-1NaCl, 3%CTAB, 2%PVP, 4% beta -mercaptoethanol), fully mix
65 DEG C of water-bath 1h afterwards, shake up once every 10-15min；
2. taking out centrifuge tube, cool down at room temperature, add 0.6mL phenol:Chloroform:Isoamyl alcohol (volume ratio 25:24:1), fully
After mixing stand a moment, the parameter of noncentricity set as 12000 × g under conditions of centrifuge 10min；
3. taking supernatant to new centrifuge tube, the chloroform of 0.6mL is added:Isoamyl alcohol (volume ratio 24:1), set after mixing
Parameter of noncentricity centrifuges 10min under conditions of being 12000 × g；
4. supernatant is taken to add the sodium acetate of 0.1mL and the isopropanol of 0.1mL-20 DEG C of precooling to new centrifuge tube, fully mix
It is even after -20 DEG C placement more than 1h, the parameter of noncentricity set as 12000 × g under conditions of centrifuge 8min；
5. abandoning supernatant, precipitation is taken to be washed 2 times with 75% ethanol, room temperature volatilization ethanol, with TE buffer or sterile water
Dissolving DNA, then (37 DEG C of water-bath 1-2h) is digested with suitable RNase A, take out and saved backup after -20 DEG C.
(3) CTAB improved methods 1
Operating procedure is with CTAB conventional methods, and NaCl concentration improves in CTAB Extraction buffers when 1. being extracted difference lies in step
To 2.50molL-1, while add 30 μ L Proteinase Ks (concentration 20mgmL-1)。
(4) CTAB improved methods 2
Operating procedure adds a small amount of absolute ethyl alcohol, Ke Yichen substantially with CTAB conventional methods, difference lies in above-mentioned steps are 3. middle
Shallow lake polysaccharide, and be beneficial to remove isolating protein.
3. above-mentioned steps are specifically changed as follows：After CTAB Extraction buffers to be utilized extracting, centrifugation, take supernatant to newly from
Heart pipe, adds 0.3mL absolute ethyl alcohols and 0.4mL phenol:Chloroform:Isoamyl alcohol (volume ratio 25:24:1) piece is stood after, fully mixing
Carve, centrifuging and taking supernatant；Subsequent operation with above-mentioned CTAB conventional methods step 4. and 5..
(5) CTAB improved methods 3
1. adding CTAB Extraction buffers after 65 DEG C of water-bath 1h, 0.5mL chloroforms are added:Isoamyl alcohol (volume ratio 24:1) take out
Carry isolating protein, the parameter of noncentricity set as 13500 × g under conditions of centrifuge 10min, take supernatant；
2. the isopropanol for adding 0.6mL-20 DEG C of precooling is used for precipitate nucleic acids, fully after mixing the parameter of noncentricity that sets as
10min is centrifuged under conditions of 13500 × g, abandons supernatant；
3. precipitation uses 0.8mL 1molL-1NaCl dissolving DNAs, add 0.8mL phenol:Chloroform (volume ratio 1:1), fully
10min is centrifuged under conditions of the parameter of noncentricity set is 13000 × g after mixing, takes supernatant；
4. add 0.8mL chloroforms:Isoamyl alcohol (volume ratio 24:1), fully mix after the parameter of noncentricity set as
8min is centrifuged under conditions of 13000 × g, takes supernatant；
5. the absolute ethyl alcohol for adding 0.8mL precoolings mixes removes impurity again, in the parameter of noncentricity set as 13500 × g
Under conditions of centrifuge 10min, abandon supernatant；
6. precipitation is washed 1 time with 70% ethanol, room temperature volatilization ethanol, subsequent operation is the same as above-mentioned CTAB conventional methods step
⑤。
(6) SDS methods
Step is as follows：
1. add 1mL Extraction buffers (100mmolL-1Tris-HCl pH 8.0、50mmol·L-1EDTA pH
8.0、0.5mol·L-1NaCl, 2% β mercaptoethanols), 0.5mL 10%SDS, 65 DEG C of water-baths after fully mixing are added after mixing
60min, during which shakes up once every 10-15min；
2. add 1mL 5molL-1Sodium acetate, ice bath 30min after mixing, in the parameter of noncentricity set as 12000 × g's
Under the conditions of centrifuge 10min；Subsequent operation with above-mentioned CTAB conventional methods step 3., 4. and 5..
(7) SDS associated proteins enzymatic lysis method
Step is as follows：Add the Extraction buffer (10mmolL of 50 DEG C of preheatings of 1mL-1Tris-HCl pH 8.0、
100mmol·L-1EDTA pH 8.0,0.5%SDS, 75mgL-1Proteinase K), 50 DEG C of water-bath 2h after fully mixing, every
10-15min shakes up once；Subsequent operation with above-mentioned CTAB conventional methods step 2., 3., 4. and 5..
(8) urea method is improved
Step is as follows：
1. add 1mL Extraction buffers (7molL-1Urea, 0.3molL-1NaCl、0.034mol·L-1Dodecyl
Sodium sarcosinate, 50mmolL-1Tris-HCl pH 8.0、20mmol·L-1EDTA pH 8.0,2% β mercaptoethanols), fully
65 DEG C of water-bath 45min, during which shake up once every 10-15min after mixing；2. in the parameter of noncentricity set as the bar of 13000 × g
10min is centrifuged under part, takes supernatant to add 100 μ L 10molL-1Ammonium acetate, adds the absolute ethyl alcohol of 2 times of volumes after mixing,
After mixing 10min is centrifuged under conditions of the parameter of noncentricity set is 13000 × g；
Subsequent operation with above-mentioned CTAB conventional methods step 2., 3., 4. and 5..
The quality inspection of banana genome DNA
(1) detected through gel electrophoresis of the banana genome DNA 1. extracted by above-mentioned various methods
The 3 μ L of banana genome DNA sample by above-mentioned various method extractions are taken in 1.0% Ago-Gel (bromine containing EB-
Change second ingot) in carry out electrophoresis respectively, observe, take pictures under gel imaging system.
In Fig. 1 (Fig. 1-1, Fig. 1-2, Fig. 1-3 and Fig. 1-4), 1：Swimming using the genomic DNA of banana pulp as sample
Road；2：Swimming lane using the genomic DNA of Banana peel as sample；3：Swimming lane using the genomic DNA of Banana Root as sample；4：With
The genomic DNA of banana caulo is the swimming lane of sample；5：Swimming lane using the genomic DNA of Banana Leaf as sample；M：Maker
(10000)；A:CTAB conventional methods；B:CTAB improved methods 1；C:CTAB improved methods 3；D:CTAB improved methods 2；E:SDS methods；F:SDS
Associated proteins enzymatic lysis method；G:Improve urea method；H:Benzyl chloride method.As shown in fig. 1, (CTAB changes A (CTAB conventional methods) with B
Good method 1) under gel electrophoresis figure it is similar, do not occur the band of genomic DNA in banana pulp and peel sample, band is unclear in root
Clear, in leaf with there is more visible band in only false stem；Gel electrophoresis figure class under C (CTAB improved methods 3) and D (CTAB improved methods 2)
Seemingly, do not occur the band of genomic DNA in Banana peel sample, more visible genomic DNA occur in the other tissues of banana
Band；E (SDS methods) is similar with gel electrophoresis figure under F (SDS associated proteins enzymatic lysises method), in banana pulp and peel sample not
There is the band of genomic DNA, and the band for occurring genomic DNA in the other tissues of banana is unintelligible；G (improvement urea method)
It is similar with gel electrophoresis figure under H (Benzyl chloride method), pulp, pericarp, root, false stem and the leaf of the banana of H (Benzyl chloride method) extractions
In have clearly main band, and band neatly becomes clear, be hardly degraded, but under G (improvement urea method), Banana peel
Do not occur the band of genomic DNA in sample.As described above, only H (Benzyl chloride method) can extract the genome during banana is respectively organized
DNA, and main band clearly become clear it is visible, i.e. this method extraction DNA integralities preferably, quality it is higher.
2. the detected through gel electrophoresis of the banana genome DNA by the Benzyl chloride method extraction for adding various concentrations benzyl chloride
The 3 μ L of banana genome DNA sample of the Benzyl chloride method extraction by different volumes benzyl chloride are taken to be coagulated in 1.0% agarose
Electrophoresis is carried out respectively in glue (ethidium bromide containing EB-), is observed, is taken pictures under gel imaging system.
In fig. 2,1：Swimming lane using the genomic DNA of banana pulp as sample；2：Using the genomic DNA of Banana peel as
The swimming lane of sample；3：Swimming lane using the genomic DNA of Banana Root as sample；4：Using the genomic DNA of banana caulo as sample
Swimming lane；5：Swimming lane using the genomic DNA of Banana Leaf as sample；M：Maker(10000)；C1：The benzyl chloride of 400 μ L benzyl chlorides
Method；C2：The Benzyl chloride method of 200 μ L benzyl chlorides；C3：The Benzyl chloride method of 600 μ L benzyl chlorides；C4：The benzyl chloride of 800 μ L benzyl chlorides
Method.As shown in Figure 2, under C4, band substantially pulls up lame with the band in other swimming lanes in 1 swimming lane (pulp), and band
Fuzzy, this is probably to contain more protein and phenols in the pulp genomic DNA；Under C1, C2 and C3, band situation
Substantially it is similar, but under C1 and C2, band is very light in swimming lane 2, and in C3, the band of each swimming lane is than more visible, neat.To sum up institute
Show, C3 (Benzyl chloride methods of 600 μ L benzyl chlorides) is most suitable for extracting the genomic DNA during banana is respectively organized.
(2) absorbance detection
Detected using nucleic acid-protein analyzer (Eppendorf BioPhotometer Plus) and carried by above-mentioned various methods
Mass concentration (quality of the genomic DNA extracted in unit sample, the unit of the banana genome DNA sample taken：μg/mL)
And absorption value A260 and A280 at wavelength 260nm, 280nm, A260/A280 is calculated to judge the purity of DNA.Table 1
The mass concentration (μ g/mL) of distinct methods extraction each tissue DNA of banana is shown.Table 2 shows different DNA extraction method extraction bananas
The A260/A280 values of each tissue DNA.Table 3 shows to add various concentrations benzyl chloride extraction each tissue DNA of banana in Benzyl chloride method
Mass concentration (μ g/mL), A260/A280 values.
The A260/A280 ratios of pure dna are 1.8, if A260/A280 ratios between 1.7~1.9, show that DNA's is pure
Degree is higher；If ratio 1.5 and it is following if represent to contain more than 50% protein in DNA solution, i.e. DNA is subject to albumen (fragrance
Race) or aldehydes matter pollution.
Clearly visible Benzyl chloride method (600 μ L) is no matter in the leaf, false stem and root of banana in table 1, or in pericarp and
The mass concentration of the genomic DNA extracted in pulp is above the genomic DNA extracted in other methods.
As shown in table 2, only CTAB improved methods 2 and Benzyl chloride method (600 μ L) can respectively be organized from banana (leaf, false stem, root,
Pericarp and pulp) in extract genomic DNA；And only the A260/A280 ratios of Benzyl chloride method (600 μ L) 1.7~
Between 1.9, as described above, only Benzyl chloride method (600 μ L) extracts the high base of purity in respectively being organized from banana in the above method
Because of a group DNA.
As shown in table 3, the genomic DNA that the Benzyl chloride method of 600 μ L benzyl chlorides extracts in Banana Leaf, false stem, root
Mass concentration is above the Benzyl chloride method of other concentration, and A260/A280 ratios are between 1.7~1.9；Although 600 μ L
The mass concentration for the genomic DNA that the Benzyl chloride method of benzyl chloride extracts in banana pulp is less than the benzyl chloride of 800 μ L benzyl chlorides
Method, but the A260/A280 ratios of the Benzyl chloride method of 600 μ L benzyl chlorides are higher than the A260/ of the Benzyl chloride method of 800 μ L benzyl chlorides
A280 ratios, and less than 1.8, in addition, the genome that the Benzyl chloride method of 600 μ L benzyl chlorides extracts in Banana peel, pulp
The mass concentration of DNA obviously higher than 200 μ L, the benzyl chloride of 400 μ L Benzyl chloride method in the quality of genomic DNA extracted it is dense
Degree.
In conclusion in the case where considering genomic DNA mass concentration and purity, the chlorination of 600 μ L benzyl chlorides
Benzyl method is the best practice for the genomic DNA for extracting banana different tissues.
The mass concentration contrast of 1 distinct methods of table extraction each tissue DNA of banana
The A260/A280 values contrast of the different DNA extraction method extraction each tissue DNAs of banana of table 2
The mass concentration contrast of various concentrations benzyl chloride extraction each tissue DNA of banana is added in 3 Benzyl chloride method of table
(3) PCR amplification detects
Respectively from banana variety：Calcutta 4；Brazilian any of several broadleaf plants (fragrant tooth any of several broadleaf plants class)；Mbwazirume (East African altiplano banana)；
Mbi Egome1(Plantain)；Cross bee saage；Dongguan plantain (plantain class)；Wide powder 1 (dwarf banana class)；Sampled in tribute any of several broadleaf plants, then
By all samples rapidly with 95% alcohol washes, be respectively placed in -80 DEG C of ultra low temperature freezers and save backup.
The above-mentioned each sample of 0.5g is taken, adds grind into powder after appropriate liquid nitrogen, is gone to rapidly in 2mL centrifuge tubes, Ran Houfen
An not Benzyl chloride method (600 μ L benzyl chlorides of addition) extraction genomic DNA.
Using above-mentioned genomic DNA as template, PCR expansions are carried out using banana the Internal Transcribed Spacer universal primer ITS4/ITSL
Increase, product EB after 1% agarose gel electrophoresis is dyed, and is observed, is taken pictures under ultraviolet light.
In figure 3, M：The swimming lane of Maker (2000)；Reference numeral 1,2,3,4,5,6,7 and 8 represents banana product respectively
Kind of Calcutta 4, Brazilian any of several broadleaf plants (fragrant tooth any of several broadleaf plants class), Mbwazirume (East African altiplano banana), Mbi Egome1 (Plantain),
Cross the swimming lane of the PCR product of the genomic DNA of bee saage, Dongguan plantain (plantain class), wide powder 1 (dwarf banana class) and tribute any of several broadleaf plants.Such as
Shown in Fig. 3, electrophoretic band is high-visible, is the band of single 700bp or so size, meets banana germplasm ITS spies
There is fragment, it can be seen that, the DNA extracted by Benzyl chloride method (600 μ L benzyl chlorides of addition) meets the demand that PCR is operated.
In conclusion the genomic DNA of banana different tissues, Er Qiesuo can be simply and quickly extracted using Benzyl chloride method
The genomic DNA integrality of extraction is good, purity is high, suitable for the demand of PCR operations；Other method phases with extracting genomic DNA
Than for extracting the genomic DNA of Banana peel, pulp, the Benzyl chloride method effect of 600 μ L benzyl chlorides especially highlights.
Although having been presented for the specific embodiment of the present invention herein, it will be appreciated by those of skill in the art that
Without departing from the spirit of the invention, the embodiments herein can be changed.Above-described embodiment is only exemplary, no
Restriction that should be using the embodiments herein as interest field of the present invention.
Claims (6)
1. a kind of method of genomic DNA suitable for rapid extraction banana different tissues, it is characterised in that including following step
Suddenly：
Step 1：Banana Tissue sample is taken, liquid nitrogen grinding is added into powder, goes in centrifuge tube rapidly；
Step 2：Extraction buffer, 10%SDS and benzyl chloride are added in the Banana Tissue sample of the step 1, is filled
Divide water-bath after mixing；
Step 3：Sodium acetate ice bath is added in the mixed liquor obtained through the step 2, is then centrifuged for；
Step 4：The supernatant learnt from else's experience obtained by the step 3 adds the isopropanol of precooling to a clean centrifuge tube, after mixing from
The heart, abandons supernatant；
Step 5：The precipitation that the step 4 of learning from else's experience obtains is washed with 75% ethanol, and room temperature volatilization ethanol, then adds TE
Buffer or sterile water, then digested with suitable RNase A, take out and saved backup after -20 DEG C；
The amount of Banana Tissue sample described in the step 1 is 0.5g；Extraction buffer described in the step 2 is 1ml, institute
State 10%SDS be 150 μ l, the benzyl chloride be 600 μ l；It is 450 μ l that the sodium acetate is added in the step 3.
2. a kind of method of genomic DNA suitable for rapid extraction banana different tissues as claimed in claim 1, its feature
It is, the Extraction buffer includes the 100mmolL of pH 9.0-1The 40mmolL of Tris-HCl, pH 8.0-1EDTA。
3. such as claim 1, a kind of side of genomic DNA suitable for rapid extraction banana different tissues of 2 any one of them
Method, it is characterised in that bath temperature is 65 DEG C in the step 2, water bath time 45min.
4. such as claim 1, a kind of side of genomic DNA suitable for rapid extraction banana different tissues of 2 any one of them
Method, it is characterised in that the time of mixed liquor described in sodium acetate ice bath described in the step 3 is 15min, and 13000 × g
Centrifuge 8min.
5. such as claim 1, a kind of side of genomic DNA suitable for rapid extraction banana different tissues of 2 any one of them
Method, it is characterised in that in the step 4,13500 × g centrifugations 10min.
6. such as claim 1, a kind of side of genomic DNA suitable for rapid extraction banana different tissues of 2 any one of them
Method, it is characterised in that in the step 5, described 75% ethanol washs the number of the precipitation as twice.
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