CN109439650A - A kind of extracting method of Agrobacterium plasmid DNA - Google Patents
A kind of extracting method of Agrobacterium plasmid DNA Download PDFInfo
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- CN109439650A CN109439650A CN201811175551.6A CN201811175551A CN109439650A CN 109439650 A CN109439650 A CN 109439650A CN 201811175551 A CN201811175551 A CN 201811175551A CN 109439650 A CN109439650 A CN 109439650A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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 discloses the extracting methods of Agrobacterium plasmid DNA a kind of, include the following steps: the bacterium solution culture of (1) Agrobacterium；(2) microorganism collection；(3) agrobatcerium cell ruptures；(4) extraction of Plasmid DNA.The present invention provides the extracting methods of Agrobacterium plasmid DNA a kind of, and methodological science is reasonable, shorten the time of extraction, improve the efficiency of extraction, improve the purity and content of Plasmid DNA, and highly-safe, have good marketing application value.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of extracting method of Agrobacterium plasmid DNA.
Background technique
The extraction and detection of Agrobacterium plasmid DNA is required test in plant genetic engineering research, and in molecule
A routine techniques in biological test.Agrobacterium transformation system is a kind of natural Transformation Systems.Agrobacterium is divided into root
Tumor Agrobacterium and agrobacterium rhizogenes.Contain tumor inducing (T) plasmid in agrobacterium tumefaciens, contains transferable DNA(T- on Ti-plasmids
DNA) area, toxicity area (area Vir) and crown gall alkali metabolic gene code area.The both ends T-DNA are the repetitive sequences of two 25bp, point
Also known as it is left margin and right margin, is that auxin and basic element of cell division synthesis gene and crown gall alkali close between two border sequences
At gene.Also contain multiple gene sections, such as FiM, FirB, FirC, VirD., VirE, VirG, VirH, Mei Geji in the area Vir
Because section all contains multiple genes.When plant comes to harm, the juice containing phenolic compound, these phenolic compounds one are secreted
For aspect by chromosome virulent gene, the chemotaxis of the mediations such as chvA, chvB promotes Agrobacterium mobile simultaneously to plant injury
It is attached to surface of Plant callus cell；On the other hand it is then identified on Ti-plasmids by the bi-component regulating system that VirA and VirG are formed,
To induce the expression of other Vir.VirD1 and VirD2 collective effect is started to cut production to left margin from T-DNA right margin
Life-T-DNA is single-stranded, and is transferred to outside Agrobacterium with the other expression protein bindings of Vir at complex, arrives subsequently into cell
In up to nucleus and it is integrated on cell chromosome.
Genetically modified plants are increasingly in the research and application of the every field such as the modern industry, pharmaceutical sector, particularly agricultural
The mankind are paid attention to.Plasmid conversion is carried out to infecting for plant, organ, tissue and cell by Agrobacterium, is by external source base
Because importing one of the important method of purpose plant.Foreign gene is artificially imported in natural plants, from inhereditary material level on pair
Plant is purposefully transformed, and thus to obtain genetically modified plants, character will change according to the direction for being conducive to people's needs
Become.As the pest-resistant cotton of transgenosis makes cotton that can automatically generate resistance in vivo during the growth process by the transformation of people
The toxin of pest, to achieve the purpose that self-protection, reduce Pesticide use increasing both production and income.
In order to which foreign gene is preferably imported purpose plant, need to develop a kind of method for extracting Agrobacterium plasmid DNA.
The program that the extracting method of conventional Agrobacterium plasmid DNA is usually directed to is more complicated, and how more expensive used reagent is, and
And the recovery rate of Agrobacterium is very low, significantly limits the development of genetic engineering.
Summary of the invention
The purpose of the present invention is being directed to existing problem, the extracting method of Agrobacterium plasmid DNA a kind of, method are provided
It is scientific and reasonable, the time of extraction is shortened, the efficiency of extraction is improved, improves the purity and content of Plasmid DNA, and safe
Property it is high, there is good marketing application value.
The present invention is achieved by the following technical solutions:
A kind of extracting method of Agrobacterium plasmid DNA, includes the following steps:
(1) Agrobacterium bacterium solution culture:
Agrobacterium single colonie after picking conversion puts into 4 ~ 5mL in the YEB fluid nutrient medium of kanamycins, then will
YEB culture medium, which is placed in shaking table, carries out shake culture, and ultrasonication is carried out while shaken cultivation；
(2) microorganism collection:
Until the OD value of bacterium solution in step (1) reaches 1.2 ~ 1.3, bacterium solution is placed in a centrifuge and carries out substep centrifugal treating,
Be that 1 ~ 2min is centrifuged with the revolving speed of 6000 ~ 7000rpm first, abandon supernatant, then with the revolving speed centrifugation 1 of 3000 ~ 4000rpm ~
2min abandons supernatant and obtains thallus；
(3) agrobatcerium cell ruptures:
Measurement 320 ~ 350mL premixed liquid is placed in step (2) and is equipped in the centrifuge tube of thallus, and 0.9 ~ 1mL lysozyme is then added, then
After carrying out 30 ~ 40s of homogenization with sterile homogenizer, the centrifuge tube that turns upside down until thallus is completely dissolved, be finally placed in -2 ~
4 ~ 6min of placement is carried out in 0 DEG C of environment；
(4) extraction of Plasmid DNA:
A. the cell solution after rupture in step (3) is placed in a centrifuge carry out centrifugal treating, the revolving speed of centrifuge is
After 11000 ~ 13000rpm, 7 ~ 8min of centrifugal treating, supernatant is taken to be placed in new sterile centrifugation tube, then to new sterile centrifugation
The dehydrated alcohol being pre-chilled in equal volume with supernatant is added in pipe, mixing is placed on 3 ~ 5min of placement in -50 ~ -40 DEG C of environment；
B. the mixed liquor operated in a being placed in a centrifuge carry out centrifugal treating, the revolving speed of centrifuge is 11000 ~
13000rpm after being centrifuged 4 ~ 5min, abandons supernatant；
C. it measures 60 ~ 80mL TE Buffer solution to be placed in the centrifuge tube of operation b, flicks the bottom of centrifuge tube, until precipitating
It is completely dissolved.
Further, in the step (1) shake culture condition are as follows: the revolving speed of shaking table is 160 ~ 180rpm, in shaking table
Temperature control be 28 ~ 32 DEG C.
Further, the frequency of ultrasonic wave is 34 ~ 38kHz in the step (1).
Further, the operating pressure of homogenizer is 30 ~ 40MPa in the step (3).
Further, the preparation method of premixed liquid is in the step (3): by Solution I, Solution II according to body
For product than being that 1:1.6 ~ 1.8 are collectively disposed in centrifuge tube, mixing, which is placed in 2 ~ 4 DEG C of environment, carries out 10 ~ 14min of placement, then measures
It takes the Solution III of II total volume 30 ~ 40% of Solution I and Solution to put into centrifuge tube, shakes up and be placed on -1 ~ 2 DEG C
Environment in place it is spare；
Further, each ingredient and corresponding parts by weight in the Solution I are as follows: 6 ~ 7 parts of 45 ~ 55mmol/L glucose, 20 ~
2 ~ 3 parts, 1 ~ 2 part of 8 ~ 12mmol/L ethylenediamine tetra-acetic acid of 30mmol/L tri- (methylol) aminomethane.
Further, each ingredient and corresponding parts by weight in the Solution II are as follows: 0.2 ~ 0.3mol/L sodium hydroxide 5 ~ 6
Part, 5 ~ 6 parts of 0.9 ~ 1.1% lauryl sodium sulfate.
Further, each ingredient and corresponding parts by weight in the Solution III are as follows: 6 ~ 7 parts of 1 ~ 2mol/L potassium acetate, ice
0.3 ~ 0.4 part of acetic acid, 3 ~ 4 parts of distilled water.
Further, each ingredient and corresponding parts by weight in step (4) the operation c are as follows: 8 ~ 12mmol/L tri- (methylol)
4 ~ 5 parts of aminomethane, 3 ~ 4 parts of 0.1 ~ 0.2mmol/L ethylenediamine tetra-acetic acid.
The present invention has done very big improvement on the basis of existing Agrobacterium plasmid DNA extraction method, is agriculture bar first
Bacterium bacterium solution culture, earthquake culture while, carry out ultrasonication, and ultrasonication can accelerate molecular motion velocities, accelerate
The breeding of thallus, and thallus efficient expression plasmid DNA can also be promoted, ultrasonication is a kind of now relatively common side
Formula, but during bacterium solution culture, the underfrequency of ultrasonic wave, facilitation effect is general, frequency is excessively high to will cause bacterium solution
Cell rupture causes a large amount of dead bacterium, brings detrimental effect to the culture of bacterium solution, applicant a large amount of it was found that,
When the frequency of ultrasonic wave is lower than 38kHz, with the raising of frequency, treatment effect is better, therefore the frequency of ultrasonic wave of the present invention
Rate is set as 34 ~ 38kHz, while accelerating thallus breeding, promotes thallus efficient expression plasmid DNA.In the mistake of microorganism collection
Cheng Zhong is carried out substep centrifugation, is first centrifuged with higher rotation speed, under the action of higher rotation speed, thallus is almost deposited on
The bottom of centrifuge tube, discards supernatant, and abandons supernatant processing only by primary centrifugation, can have thallus and lose serious phenomenon, because
This present invention has carried out a low-speed centrifugal again, and the thallus in supernatant is precipitated completely.During agrobatcerium cell rupture,
Using premixed liquid, a large amount of time is saved, and has carried out homogenization during rupture, at homogeneous appropriate
Reason promotes the dissolution of cell Dissolve things inside, further speeds up the speed and efficiency of extraction of plasmid DNA.
The present invention has the advantage that compared with prior art
The present invention provides the extracting methods of Agrobacterium plasmid DNA a kind of, and methodological science is reasonable, shorten the time of extraction, mention
The high efficiency extracted, improves the purity and content of Plasmid DNA, and highly-safe, has good marketing application
Value.
Specific embodiment
Embodiment 1
A kind of extracting method of Agrobacterium plasmid DNA, includes the following steps:
(1) Agrobacterium bacterium solution culture:
Agrobacterium single colonie after picking conversion is put into 4mL and is had in the YEB fluid nutrient medium of kanamycins, then by YEB
Culture medium, which is placed in shaking table, carries out shake culture, and ultrasonication is carried out while shaken cultivation；
(2) microorganism collection:
Until the OD value of bacterium solution in step (1) reaches 1.2, bacterium solution is placed in a centrifuge and carries out substep centrifugal treating, first
It is that 1min is centrifuged with the revolving speed of 6000rpm, abandons supernatant, 1min is then centrifuged with the revolving speed of 3000rpm, abandons supernatant and obtain thallus；
(3) agrobatcerium cell ruptures:
It measures 320mL premixed liquid and is placed in step (2) equipped in the centrifuge tube of thallus, 0.9mL lysozyme is then added, then with sterile
Homogenizer carry out homogenization 30s after, turn upside down centrifuge tube until thallus be completely dissolved, be finally placed in -2 DEG C of environment into
Row places 4min；
(4) extraction of Plasmid DNA:
A. the cell solution after rupture in step (3) is placed in a centrifuge carry out centrifugal treating, the revolving speed of centrifuge is
After 11000rpm, centrifugal treating 7min, supernatant is taken to be placed in new sterile centrifugation tube, is then added into new sterile centrifugation tube
The dehydrated alcohol being pre-chilled in equal volume with supernatant, mixing, which is placed in -50 DEG C of environment, places 3min；
B. the mixed liquor operated in a is placed in a centrifuge carry out centrifugal treating, the revolving speed of centrifuge is 11000rpm, centrifugation
After 4min, supernatant is abandoned；
C. it measures 60mL TE Buffer solution to be placed in the centrifuge tube of operation b, the bottom of centrifuge tube is flicked, until having precipitated
Fully dissolved.
Further, in the step (1) shake culture condition are as follows: the revolving speed of shaking table is 160rpm, the temperature in shaking table
Degree control is 28 DEG C.
Further, the frequency of ultrasonic wave is 34kHz in the step (1).
Further, the operating pressure of homogenizer is 30MPa in the step (3).
Further, the preparation method of premixed liquid is in the step (3): by Solution I, Solution II according to body
For product than being that 1:1.6 is collectively disposed in centrifuge tube, mixing, which is placed in 2 DEG C of environment, carries out placement 10min, then measures Solution
The Solution III of I and Solution, II total volume 30% is put into centrifuge tube, shake up placed in the environment for be placed on -1 DEG C it is standby
With；
Further, each ingredient and corresponding parts by weight in the Solution I are as follows: 6 parts of 45mmol/L glucose, 20mmol/L tri-
2 parts of (methylol) aminomethane, 1 part of 8mmol/L ethylenediamine tetra-acetic acid.
Further, each ingredient and corresponding parts by weight in the Solution II are as follows: 5 parts of 0.2mol/L sodium hydroxide,
0.9% 5 parts of lauryl sodium sulfate.
Further, each ingredient and corresponding parts by weight in the Solution III are as follows: 6 parts of 1mol/L potassium acetate, glacial acetic acid
0.3 part, 3 parts of distilled water.
Further, each ingredient and corresponding parts by weight in step (4) the operation c are as follows: 8mmol/L tri- (methylol) amino
4 parts of methane, 3 parts of 0.1mmol/L ethylenediamine tetra-acetic acid.
Embodiment 2
A kind of extracting method of Agrobacterium plasmid DNA, includes the following steps:
(1) Agrobacterium bacterium solution culture:
Agrobacterium single colonie after picking conversion puts into 4.5mL in the YEB fluid nutrient medium of kanamycins, then will
YEB culture medium, which is placed in shaking table, carries out shake culture, and ultrasonication is carried out while shaken cultivation；
(2) microorganism collection:
Until the OD value of bacterium solution in step (1) reaches 1.25, bacterium solution is placed in a centrifuge and carries out substep centrifugal treating, first
It is that 1.5min is centrifuged with the revolving speed of 6500rpm, abandons supernatant, 1.5min is then centrifuged with the revolving speed of 3500rpm, abandons supernatant and obtain bacterium
Body；
(3) agrobatcerium cell ruptures:
It measures 335mL premixed liquid and is placed in step (2) equipped in the centrifuge tube of thallus, 0.95mL lysozyme is then added, then use nothing
After the homogenizer of bacterium carries out homogenization 35s, the centrifuge tube that turns upside down finally is placed in -1 DEG C of environment until thallus is completely dissolved
Carry out placement 5min；
(4) extraction of Plasmid DNA:
A. the cell solution after rupture in step (3) is placed in a centrifuge carry out centrifugal treating, the revolving speed of centrifuge is
After 12000rpm, centrifugal treating 7.5min, supernatant is taken to be placed in new sterile centrifugation tube, then added into new sterile centrifugation tube
Enter the dehydrated alcohol being pre-chilled in equal volume with supernatant, mixing, which is placed in -45 DEG C of environment, places 4min；
B. the mixed liquor operated in a is placed in a centrifuge carry out centrifugal treating, the revolving speed of centrifuge is 12000rpm, centrifugation
After 4.5min, supernatant is abandoned；
C. it measures 70mL TE Buffer solution to be placed in the centrifuge tube of operation b, the bottom of centrifuge tube is flicked, until having precipitated
Fully dissolved.
Further, in the step (1) shake culture condition are as follows: the revolving speed of shaking table is 170rpm, the temperature in shaking table
Degree control is 30 DEG C.
Further, the frequency of ultrasonic wave is 36kHz in the step (1).
Further, the operating pressure of homogenizer is 35MPa in the step (3).
Further, the preparation method of premixed liquid is in the step (3): by Solution I, Solution II according to body
For product than being that 1:1.7 is collectively disposed in centrifuge tube, mixing, which is placed in 3 DEG C of environment, carries out placement 12min, then measures Solution
The Solution III of I and Solution, II total volume 35% is put into centrifuge tube, shake up placed in the environment for be placed on 0 DEG C it is spare；
Further, each ingredient and corresponding parts by weight in the Solution I are as follows: 6.5 parts of 50mmol/L glucose, 25mmol/L
Three 2.5 parts of (methylol) aminomethanes, 1.5 parts of 10mmol/L ethylenediamine tetra-acetic acid.
Further, each ingredient and corresponding parts by weight in the Solution II are as follows: 5.5 parts of 0.25mol/L sodium hydroxide,
1% 5.5 parts of lauryl sodium sulfate.
Further, each ingredient and corresponding parts by weight in the Solution III are as follows: 6.5 parts of 1.5mol/L potassium acetate, ice
0.35 part of acetic acid, 3.5 parts of distilled water.
Further, each ingredient and corresponding parts by weight in step (4) the operation c are as follows: 10mmol/L tri- (methylol) ammonia
4.5 parts of methylmethane, 3.5 parts of 0.15mmol/L ethylenediamine tetra-acetic acid.
Embodiment 3
A kind of extracting method of Agrobacterium plasmid DNA, includes the following steps:
(1) Agrobacterium bacterium solution culture:
Agrobacterium single colonie after picking conversion is put into 5mL and is had in the YEB fluid nutrient medium of kanamycins, then by YEB
Culture medium, which is placed in shaking table, carries out shake culture, and ultrasonication is carried out while shaken cultivation；
(2) microorganism collection:
Until the OD value of bacterium solution in step (1) reaches 1.3, bacterium solution is placed in a centrifuge and carries out substep centrifugal treating, first
It is that 2min is centrifuged with the revolving speed of 7000rpm, abandons supernatant, 2min is then centrifuged with the revolving speed of 4000rpm, abandons supernatant and obtain thallus；
(3) agrobatcerium cell ruptures:
It measures 350mL premixed liquid and is placed in step (2) equipped in the centrifuge tube of thallus, 1mL lysozyme is then added, then with sterile
Homogenizer carry out homogenization 40s after, turn upside down centrifuge tube until thallus be completely dissolved, be finally placed in 0 DEG C of environment and carry out
Place 6min；
(4) extraction of Plasmid DNA:
A. the cell solution after rupture in step (3) is placed in a centrifuge carry out centrifugal treating, the revolving speed of centrifuge is
After 13000rpm, centrifugal treating 8min, supernatant is taken to be placed in new sterile centrifugation tube, is then added into new sterile centrifugation tube
The dehydrated alcohol being pre-chilled in equal volume with supernatant, mixing, which is placed in -40 DEG C of environment, places 5min；
B. the mixed liquor operated in a is placed in a centrifuge carry out centrifugal treating, the revolving speed of centrifuge is 13000rpm, centrifugation
After 5min, supernatant is abandoned；
C. it measures 80mLTE Buffer solution to be placed in the centrifuge tube of operation b, flicks the bottom of centrifuge tube, until precipitating is complete
Dissolution.
Further, in the step (1) shake culture condition are as follows: the revolving speed of shaking table is 180rpm, the temperature in shaking table
Degree control is 32 DEG C.
Further, the frequency of ultrasonic wave is 38kHz in the step (1).
Further, the operating pressure of homogenizer is 40MPa in the step (3).
Further, the preparation method of premixed liquid is in the step (3): by Solution I, Solution II according to body
For product than being that 1:1.8 is collectively disposed in centrifuge tube, mixing, which is placed in 4 DEG C of environment, carries out placement 14min, then measures Solution
The Solution III of I and Solution, II total volume 40% is put into centrifuge tube, shake up placed in the environment for be placed on 2 DEG C it is spare；
Further, each ingredient and corresponding parts by weight in the Solution I are as follows: 7 parts of 55mmol/L glucose, 30mmol/L tri-
3 parts of (methylol) aminomethane, 2 parts of 12mmol/L ethylenediamine tetra-acetic acid.
Further, each ingredient and corresponding parts by weight in the Solution II are as follows: 6 parts of 0.3mol/L sodium hydroxide,
1.1% 6 parts of lauryl sodium sulfate.
Further, each ingredient and corresponding parts by weight in the Solution III are as follows: 7 parts of 2mol/L potassium acetate, glacial acetic acid
0.4 part, 4 parts of distilled water.
Further, each ingredient and corresponding parts by weight in step (4) the operation c are as follows: 12mmol/L tri- (methylol) ammonia
5 parts of methylmethane, 4 parts of 0.2mmol/L ethylenediamine tetra-acetic acid.
Comparative example 1
This comparative example 1 compared with Example 2, saves the ultrasonication in step (1), and method and step in addition to this is homogeneous
Together.
Comparative example 2
This comparative example 2 compared with Example 2, saves the homogenization in step (3), and method and step in addition to this is homogeneous
Together.
Comparative example 3
This comparative example 3 changes conventional agriculture into compared with Example 2, by the whole operation that step (3) agrobatcerium cell ruptures
Bacilli-cell disruption method, method and step in addition to this are all the same.
Control group
Application No. is: a kind of kit extracting Agrobacterium plasmid DNA disclosed in 201510449485.7.
In order to compare effect of the present invention, from same plate picking conversion after Agrobacterium single colonie, respectively with implement
Example 2, comparative example 1, comparative example 2, comparative example 3, the method for control group are extracted Agrobacterium plasmid DNA, and are measured
Index of correlation, plasmid content are determined using the A value of ultraviolet specrophotometer measurement 260nm wavelength.The measurement of plasmid purity uses
The absorption ratio that ultraviolet specrophotometer measures 260nm, 280nm wavelength determines that (ratio met the quality standard should be in 1.75-
Between 1.85) row agarose gel electrophoresis of going forward side by side.
Specific experiment correlation data such as the following table 1 and
Table 1
The present invention provides the extracting method of Agrobacterium plasmid DNA a kind of it can be seen from upper table 1, methodological science is reasonable, shortens
It the time extracted, the efficiency of extraction is improved, improves the purity and content of Plasmid DNA, and highly-safe, have fine
Marketing application value.
Claims (9)
1. a kind of extracting method of Agrobacterium plasmid DNA, which comprises the steps of:
(1) Agrobacterium bacterium solution culture:
Agrobacterium single colonie after picking conversion puts into 4 ~ 5mL in the YEB fluid nutrient medium of kanamycins, then will
YEB culture medium, which is placed in shaking table, carries out shake culture, and ultrasonication is carried out while shaken cultivation；
(2) microorganism collection:
Until the OD value of bacterium solution in step (1) reaches 1.2 ~ 1.3, bacterium solution is placed in a centrifuge and carries out substep centrifugal treating,
Be that 1 ~ 2min is centrifuged with the revolving speed of 6000 ~ 7000rpm first, abandon supernatant, then with the revolving speed centrifugation 1 of 3000 ~ 4000rpm ~
2min abandons supernatant and obtains thallus；
(3) agrobatcerium cell ruptures:
Measurement 320 ~ 350mL premixed liquid is placed in step (2) and is equipped in the centrifuge tube of thallus, and 0.9 ~ 1mL lysozyme is then added, then
After carrying out 30 ~ 40s of homogenization with sterile homogenizer, the centrifuge tube that turns upside down until thallus is completely dissolved, be finally placed in -2 ~
4 ~ 6min of placement is carried out in 0 DEG C of environment；
(4) extraction of Plasmid DNA:
A. the cell solution after rupture in step (3) is placed in a centrifuge carry out centrifugal treating, the revolving speed of centrifuge is
After 11000 ~ 13000rpm, 7 ~ 8min of centrifugal treating, supernatant is taken to be placed in new sterile centrifugation tube, then to new sterile centrifugation
The dehydrated alcohol being pre-chilled in equal volume with supernatant is added in pipe, mixing is placed on 3 ~ 5min of placement in -50 ~ -40 DEG C of environment；
B. the mixed liquor operated in a being placed in a centrifuge carry out centrifugal treating, the revolving speed of centrifuge is 11000 ~
13000rpm after being centrifuged 4 ~ 5min, abandons supernatant；
C. it measures 60 ~ 80mL TE Buffer solution to be placed in the centrifuge tube of operation b, flicks the bottom of centrifuge tube, until precipitating
It is completely dissolved.
2. requiring a kind of 1 extracting method of Agrobacterium plasmid DNA according to power, which is characterized in that concussion in the step (1)
The condition of culture are as follows: the revolving speed of shaking table is 160 ~ 180rpm, and the temperature control in shaking table is 28 ~ 32 DEG C.
3. a kind of extracting method of Agrobacterium plasmid DNA according to claim 1, which is characterized in that surpass in the step (1)
The frequency of sound wave is 34 ~ 38kHz.
4. a kind of extracting method of Agrobacterium plasmid DNA according to claim 1, which is characterized in that in the step (3)
The operating pressure of matter machine is 30 ~ 40MPa.
5. a kind of extracting method of Agrobacterium plasmid DNA according to claim 1, which is characterized in that pre- in the step (3)
The preparation method of mixed liquid is: Solution I, Solution II is collectively disposed in centrifuge tube according to volume ratio for 1:1.6 ~ 1.8,
Mixing is placed in 2 ~ 4 DEG C of environment and carries out 10 ~ 14min of placement, then measure II total volume 30 of Solution I and Solution ~
40% Solution III is put into centrifuge tube, shake up placed in the environment for be placed on -1 ~ 2 DEG C it is spare.
6. a kind of extracting method of Agrobacterium plasmid DNA according to claim 5, which is characterized in that in the Solution I
Each ingredient and corresponding parts by weight are as follows: 6 ~ 7 parts of 45 ~ 55mmol/L glucose, 20 ~ 30mmol/L tri- (methylol) aminomethane 2 ~ 3
Part, 1 ~ 2 part of 8 ~ 12mmol/L ethylenediamine tetra-acetic acid.
7. a kind of extracting method of Agrobacterium plasmid DNA according to claim 5, which is characterized in that the Solution II
In each ingredient and corresponding parts by weight are as follows: 5 ~ 6 parts of 0.2 ~ 0.3mol/L sodium hydroxide, 5 ~ 6 parts of 0.9 ~ 1.1% lauryl sodium sulfate.
8. a kind of extracting method of Agrobacterium plasmid DNA according to claim 5, which is characterized in that the Solution III
In each ingredient and corresponding parts by weight are as follows: 6 ~ 7 parts of 1 ~ 2mol/L potassium acetate, 0.3 ~ 0.4 part of glacial acetic acid, 3 ~ 4 parts of distilled water.
9. a kind of extracting method of Agrobacterium plasmid DNA according to claim 1, which is characterized in that step (4) operation
Each ingredient and corresponding parts by weight in c are as follows: 4 ~ 5 parts of 8 ~ 12mmol/L tri- (methylol) aminomethane, 0.1 ~ 0.2mmol/L second two
3 ~ 4 parts of amine tetraacethyl.
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