CN107034155A - It is a kind of to be used to prepare the buffer solution of competent cell and prepare the method for competent cell - Google Patents
It is a kind of to be used to prepare the buffer solution of competent cell and prepare the method for competent cell Download PDFInfo
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Abstract
The invention discloses a kind of for preparing the buffer solution of competent cell and preparing the method for competent cell, belong to biological technical field.The buffer solution is to include piperazine 1,4 two ethyl sulfonic acids, N, N (2 ethoxy) 2 tarines, calcium chloride, the aqueous solution of potassium chloride and manganese chloride, the pH value of the buffer solution is 6.0~7.0, piperazine 1 in the buffer solution, 4 two ethyl sulfonic acids, N, N (2 ethoxy) 2 tarines, calcium chloride, the concentration of potassium chloride and manganese chloride are respectively 5~30mM, 3~20mM, 5~25mM, 50~300mM and 10~50mM.Buffer solution provided in an embodiment of the present invention, by adding N, N (2 ethoxy) 2 tarines can increase membrane passage, so that the transformation efficiency of the competent cell prepared is greatly improved, and gained competent cell can be applied to the conversion of long segment, meanwhile, preparation method provided in an embodiment of the present invention can efficiently and rapidly prepare competent cell.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of buffer solution for being used to prepare competent cell and preparation sense
By the method for state cell.
Background technology
The method of exogenous DNA transformation receptor bacterium is one of most common method in molecular biology research, and exogenous DNA includes
Plasmid and connection carrier.Exogenous DNA must be transferred to appropriate recipient bacterium and be bred, and could obtain substantial amounts of positive recombinant gram
Grand or expression target gene product.
Existing is that recipient bacterium is incubated in LB plating mediums, and picking single bacterium colony is inoculated in SOB (Super
Optimal Broth) ferment in Shake flask medium, realize the breeding of single bacterium colony, 18 DEG C of shaken cultivations to OD600It is worth for 0.6, ice
10min is bathed, 10min is centrifuged at 4 DEG C, thalline is suspended in the TB buffer solutions of precooling, ice bath 10min centrifuges 10min, bacterium at 4 DEG C
Body is suspended in the TB buffer solutions of precooling, and is added after the dimethyl sulfoxide that 1.5m concentration is 7%, ice bath 10min, obtains competence
Cell simultaneously dispenses preservation.Wherein, TB buffer solutions are to include the water of piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, calcium chloride, potassium chloride and magnesium chloride
Solution, and TB buffer solutions include：Concentration is 10mM piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, and calcium chloride that concentration is 15mM, concentration are
250mM potassium chloride and concentration is 55mM magnesium chloride.Ca in TB buffer solutions2+Effect mainly destruction cell membrane on fat
Matter array, and with many poly butyric compounds on cell membrane and poly inorganic phosphate formation compound in favor of exogenous DNA
Infiltration, then promote cell to absorb exogenous DNA by 42s heat shocks.The company that this method conversion length is more than 7000bp
When carrying body, very unobtainable positive recombinant clone.
The content of the invention
The problem of competent cell transformation efficiency is low is prepared in order to solve prior art chemical method, the embodiment of the present invention is provided
It is a kind of to be used to prepare the buffer solution of competent cell and prepare the method for competent cell.The technical scheme is as follows：
On the one hand, the embodiments of the invention provide a kind of buffer solution for being used to prepare competent cell, the buffer solution is
Including the ethyl sulfonic acids of piperazine -1,4- two, N, N- (2- ethoxys)-Tau, calcium chloride, potassium chloride and manganese chloride it is water-soluble
Liquid, the pH value of the buffer solution is piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- (2- ethoxys) -2- in 6.0~7.0, the buffer solution
Tarine, calcium chloride, the concentration of potassium chloride and manganese chloride be respectively 11~30mM, 3~20mM, 5~25mM, 50~
300mM and 10~50mM.
Specifically, piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N in the buffer solution, N- (2- ethoxys)-Tau, chlorination
The concentration of calcium, potassium chloride and manganese chloride is respectively 12~20mM, 5~15mM, 10~20mM, 150~250mM and 30~40mM,
The pH value of the buffer solution is 6.7.
More specifically, piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- (2- ethoxys)-Tau, chlorine in the buffer solution
The concentration for changing calcium, potassium chloride and manganese chloride is respectively 15mM, 10mM, 15mM, 200mM and 35mM.
On the other hand, the embodiments of the invention provide a kind of method for preparing competent cell using above-mentioned buffer solution, institute
The method of stating includes：
Monoclonal and the centrifugation of recipient bacterium are bred, the first sediment is obtained；
First sediment is resuspended with the buffer solution, the first re-suspension liquid is obtained；
It will be centrifuged after the first re-suspension liquid ice bath, remove supernatant, obtain the second sediment；
Second sediment is resuspended with the buffer solution, the second re-suspension liquid is obtained；
By second re-suspension liquid centrifugation, supernatant is removed, the 3rd sediment is obtained；
3rd sediment is placed in the mixed liquor of the buffer solution and the dimethyl sulfoxide (DMSO) and is resuspended, ice bath
After 20~30min, competent cell is obtained.
Inventor using buffer solution chemical method described in the embodiment of the present invention it was unexpectedly observed that prepare competent cell, simultaneously
Extend the 3rd sediment be placed in be resuspended in the mixed liquor of the buffer solution and the dimethyl sulfoxide (DMSO) after the ice bath time by leading to
Normal 10min is extended to after 20~30min, and gained competent cell transformation efficiency is greatly improved.
Specifically, the volume ratio 125 of first sediment and the buffer solution:(4~8).
Specifically, the volume ratio of second sediment and the buffering is (2~3):1.
Specifically, the buffer solution and the mixed liquor of dimethyl sulfoxide (DMSO) the 3rd sediment being placed in after precooling are carried out
It is resuspended, the volume ratio of the 3rd sediment, the buffer solution and the dimethyl sulfoxide (DMSO) is 200:93:7.
Further, the temperature of the mixed liquor of the buffer solution of precooling and dimethyl sulfoxide (DMSO) is 4 DEG C.
Specifically, the preparation method of the monoclonal of recipient bacterium can be prepared from method commonly known in the art,
For example the bacterium solution of the recipient bacterium is coated on plating medium, the plating medium is inverted at 37 DEG C and cultivated, directly
Extremely there is monoclonal on the plating medium.
Specifically, the monoclonal of the breeding recipient bacterium and centrifugation includes：Monoclonal described in picking is placed in SOC shaking flasks
In culture medium, shaken cultivation is to OD at 15~20 DEG C600It is worth for 0.6；The SOC Shake flask mediums after culture are placed in ice
Centrifuged after upper 10min.
The beneficial effect that technical scheme provided in an embodiment of the present invention is brought is：Buffer solution provided in an embodiment of the present invention is used
When chemical method competent cell is prepared, cation wherein in calcium chloride, potassium chloride and manganese chloride can with costimulatory receptor bacterium,
Recipient bacterium is set to become the form of acceptant exogenous DNA, piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two can stablize the cytoactive of recipient bacterium,
By adding N, N- (2- ethoxys)-Tau can increase the logical of cell membrane on the premise of cytoactive is ensured
Permeability, so that the transformation efficiency of the competent cell prepared is greatly improved, and gained competent cell can be applied to lengthy motion picture
The conversion of section；Meanwhile, preparation method provided in an embodiment of the present invention can efficiently and rapidly prepare competent cell.
Brief description of the drawings
Technical scheme in order to illustrate the embodiments of the present invention more clearly, makes required in being described below to embodiment
Accompanying drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for
For those of ordinary skill in the art, on the premise of not paying creative work, other can also be obtained according to these accompanying drawings
Accompanying drawing.
Fig. 1 is the transformation efficiency detection figure for the competent cell that the embodiment of the present invention one is provided；
Fig. 2 is the blue hickie screening experiment figure for the competent cell that the embodiment of the present invention one is provided.
Fig. 3 is the blue hickie screening experiment figure for the commercialized competent cell that the embodiment of the present invention one is provided；
Fig. 4 is the recombinant plasmid PCR testing result figures of the acquisition for the competent cell that the embodiment of the present invention one is provided, figure
In, swimming lane 1 and 2 is the white colony PCR detection knots after the random competent cell conversion for selecting the offer of the embodiment of the present invention one
Really, swimming lane 3 is AL15000 marker, and swimming lane 4 and 5 is the bacterium colony PCR detections obtained after commercialized competent cell is converted
As a result；
Fig. 5 is the double digestion testing result of the recombinant plasmid of the acquisition for the competent cell that the embodiment of the present invention one is provided,
In figure, swimming lane 1 and 6 is AL15000 and AL5000 marker respectively, and it is that the embodiment of the present invention one is provided that swimming lane 2 and 3, which is utilized,
The recombinant plasmid double digestion result that competent cell is obtained, swimming lane 4 and 5 is the competent cell restructuring matter prepared using commercialization
The double digestion result of grain；
Fig. 6 is the transformation efficiency detection figure for the competent cell that the embodiment of the present invention two is provided；
Fig. 7 is the transformation efficiency detection figure for the competent cell that the embodiment of the present invention three is provided.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with accompanying drawing to embodiment party of the present invention
Formula is described in further detail.
Agents useful for same is commercial reagent in the embodiment of the present invention, wherein, the piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- (2- hydroxyl second
Base)-Tau is biochemical reagents, is purchased from Wuhan epoch Bo Chi Science and Technology Ltd.s；Calcium chloride, potassium chloride, chlorine
It is analysis pure chemistry reagent to change manganese and potassium hydroxide, is purchased from Wuhan epoch Bo Chi Science and Technology Ltd.s.
Embodiment one
Be used to preparing the buffer solution of competent cell the embodiments of the invention provide a kind of, the buffer solution be include piperazine-
The ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- (2- ethoxys)-Tau, calcium chloride, the aqueous solution of potassium chloride and manganese chloride, the buffering
The pH value of liquid is 6.7, in the present embodiment, and 4.5354g piperazines-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, 2.1325g N are added into buffer solution,
N- (2- ethoxys)-Tau, 1.6647g calcium chloride, 14.9102g potassium chloride and 5.6656g manganese chlorides, add water
1000ml so that the piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- (2- ethoxys)-Tau, calcium chloride, potassium chloride and chlorination
The concentration of manganese is respectively 15mM, 10mM, 15mM, 200mM and 35mM, when preparing buffer solution, and buffer solution is adjusted with potassium hydroxide
PH value, and filtration sterilization is stand-by in 4 DEG C by the buffer preserving prepared, passes through the pH value that potassium hydroxide adjusts buffer solution
It ensure that the competence activity for the competent cell prepared.
The recipient bacterium selected in the present embodiment is Escherichia coli, specially bacillus coli DH 5 alpha, is purchased from TAKARA companies.
The bacterium solution of recipient bacterium is coated on plating medium, by plating medium, (composition is with reference to Molecular
Cloning:A Laboratory Mannual) incubated overnight at 37 DEG C is inverted in, until occurring Dan Ke on plating medium
It is grand；
Picking monoclonal is placed in the shaking flask equipped with Shake flask medium, and 1L shaking flasks are equipped with 500ml Shake flask mediums, and shaking flask
Culture medium is that SOC (Super Optimal broth with Catabolite repression) Shake flask medium (join by composition
According to Molecular Cloning:A Laboratory Mannual), shaken cultivation is to OD at 15 DEG C600It is worth for 0.6, and shakes
Rotating speed when swinging is 100-110rpm；
SOC Shake flask mediums after culture are placed in 10min on ice, then by SOC Shaking cultures based on centrifugation under the conditions of 4 DEG C
10min, and centrifugal rotational speed is 2500rpm, removes supernatant, obtains the first sediment；
The 16ml buffer solutions that the sediments of 500ml first are provided with the present embodiment are resuspended, and obtain the first re-suspension liquid；
First re-suspension liquid ice bath 10min is centrifuged into 10min under the conditions of 4 DEG C, and centrifugal rotational speed is 2500rpm, is gone
Clear liquid, obtains the second sediment；
The 8ml buffer solutions that the second sediments of 16ml embodiment one is provided are resuspended, and obtain the second resuspended bacterium solution；
10min will be centrifuged under the conditions of 4 DEG C of second re-suspension liquid, and centrifugal rotational speed is 2500rpm, removes supernatant, obtains the 3rd
Sediment；
The sediments of 8ml the 3rd are placed in 3.72ml buffer solutions and the 0.28ml dimethyl Asia that the embodiment after precooling one is provided
It is resuspended in the mixed liquor of sulfone, after ice bath 30min, the temperature of the buffer solution of precooling and the mixed liquor of dimethyl sulfoxide (DMSO) is 4 DEG C,
Obtain competent cell.By the competent cell prepared be stored in -70 DEG C it is stand-by.The competent cell of gained is used for length
Converted for 3000bp plasmid (plasmid is pGEM (R)-T Easy Vector), conversion process refers to Molecular
Cloning:Standard conversion flow in A Laboratory Mannual, obtains transformant 8758 altogether, and transformation efficiency is
8.758×109Cfu/ μ g, its experimental result is as shown in Figure 1, it can be seen that, the transformation efficiency of the competent cell is very high.
Carrier will be connected and be transferred to the blue hickie screening experiment of competent cell progress that embodiment one is obtained, wherein, connection
Carrier total length is 8100bp, and the connection carrier includes length and (come from for 5100 plasmid from arabidopsis PWR gene codes
Area clone) and carrier T (pGEM-T-easy is purchased in Promega companies) be formed by connecting (connect carrier preparation method be：5μ
l 2×buffer、1μl T4Ligase, 1 μ l pGem-T Easy Vector, 3 μ l arabidopsis PWR gene coding regions clone's groups
Into 10 μ l coupled reaction system, carry out staying overnight coupled reaction under the conditions of 4 DEG C, so as to obtain connecting carrier), its experimental result
As shown in Fig. 2 from Figure 2 it can be seen that it can obtain the bacterium colony of white and blueness simultaneously.Meanwhile, by commercialized competent cell
(buying in Beijing CoWin Bioscience Co., Ltd.) prepares connection carrier after the same method, and is carried out indigo plant
Hickie screening experiment, its experimental result are as shown in figure 3, as seen from Figure 3, it only obtains the bacterium colony of blueness (because picture is adjusted to
Grey picture so that the bacterium colony that blue bacterium colony appears dimmed), understood with reference to Fig. 2 and Fig. 3, connection carrier is transferred to the present invention
Embodiment one provide competent cell after can obtain white bacterium colony (positive colony), and connect carrier be transferred to it is commercialized
Then fail to access white bacterium colony after competent cell, it can be seen that, competent cell provided in an embodiment of the present invention can
Long segment plasmid is converted, and commercialized competent cell can not then convert long segment plasmid.
By the random choosing colony of above two transformant, and carry out bacterium colony PCR (Polymerase Chain Reaction,
PCR is referred to as) detection, operating method is with reference to Molecular Cloning:In A Labora tory Mannual
Standard Plate PCR testing processes, reaction system is as shown in table 1.
Primer used includes SEQ ID NO in sequence table in bacterium colony PCR reactions:Sense primer and sequence table shown in 1
Middle SEQ ID NO:Anti-sense primer shown in 2, wherein, PCR reaction reagent 2 × long Taq MasterMix are purchased from Beijing Ai De
Lay company.
Table 1 is the reaction system that PCR is expanded
| Reagent | Volume (μ l) |
| ddH2O | 8.2 |
| 2×long Taq MasterMix | 10 |
| Sense primer (10 μM) | 0.4 |
| Anti-sense primer (10 μM) | 0.4 |
| Template | 1 |
The reaction system totally 20 μ l of PCR amplifications.By the reaction system of the PCR amplifications in table 1 according to following PCR response procedures
Carry out：
PCR amplification program be：95 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extensions
5min30s, expands 35 circulations, and circulation terminates to extend 10min after 72 DEG C.
After PCR amplifications terminate, pcr amplification product is obtained, 10 μ l pcr amplification products is drawn and carries out the agar that concentration is 1%
Sugared detected through gel electrophoresis.Its testing result is as shown in figure 4, as shown in Figure 4, prepared by competence only provided in an embodiment of the present invention
The PCR testing results of transformant be positive.And transformant prepared by commercialized competent cell is then feminine gender.Thus may be used
See, competent cell provided in an embodiment of the present invention can convert long segment plasmid, and commercialized competent cell then can not
Convert long segment plasmid.
Further, the recombinant plasmid that the competent cell provided using agarose electrophoresis the embodiment of the present invention one is obtained
Progress double digestion detection after plasmid is extracted respectively with the recombinant plasmid of the acquisition of the obtained competent cell of commercialization.Specific method
For：Culture is enlarged in picking monoclonal bacterium colony, liquid medium within, then (Beijing is purchased from using the small extraction reagent kit of plasmid
Ai Delai companies), obtain recombinant plasmid.Double digestion inspection is carried out to recombinant plasmid using restriction enzyme (being purchased from NEB companies)
Survey, endonuclease reaction system is as shown in table 2
Table 2 is double digestion reaction system
By above-mentioned double digestion reaction system in 37 DEG C of digestions overnight, then carry out concentration and identified for 1% agarose electrophoresis,
Qualification result is as shown in figure 5, as shown in Figure 5, the white gram that competent cell only provided in an embodiment of the present invention is selected at random
Grand double digestion result is positive, and this further proves that competent cell provided in an embodiment of the present invention can be successfully by long segment matter
Grain is transferred to.
Embodiment two
Be used to preparing the buffer solution of competent cell the embodiments of the invention provide a kind of, the buffer solution be include piperazine-
The ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- (2- ethoxys)-Tau, calcium chloride, the aqueous solution of potassium chloride and manganese chloride, the buffering
Liquid pH value is 6.0, in the present embodiment, and 3.326g piperazines-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, 0.6398g N, N- are added into 1L buffer solutions
(2- ethoxys)-Tau, 0.5549g calcium chloride, 3.7276g potassium chloride and 1.6187g manganese chlorides, add water
1000ml so that the piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- (2- ethoxys)-Tau, calcium chloride, potassium chloride and chlorination
The concentration of manganese is respectively 11mM, 3mM, 5mM, 50mM and 10mM.The buffer preserving prepared is stand-by in 4 DEG C.
The recipient bacterium selected in the present embodiment is Escherichia coli, specially bacillus coli DH 5 alpha.
The bacterium solution of recipient bacterium is coated on plating medium, plating medium incubated overnight at 37 DEG C is inverted in, directly
Extremely occurs monoclonal on plating medium；
Picking monoclonal is placed in the shaking flask equipped with SOC Shake flask mediums, and 1L shaking flasks are equipped with 500ml SOC Shaking cultures
Base, shaken cultivation is to OD at 20 DEG C600It is worth for 0.6, and rotating speed during vibration is 100-110rpm；
SOC Shake flask mediums after culture are placed in 10min on ice, then by SOC Shaking cultures based on centrifugation under the conditions of 4 DEG C
10min, and centrifugal rotational speed is 2500rpm, removes supernatant, obtains the first sediment；
The 32ml buffer solutions that the first sediments of 500ml embodiment two is provided are resuspended, and obtain the first re-suspension liquid；
First re-suspension liquid ice bath 10min is centrifuged into 10min under the conditions of 4 DEG C, and centrifugal rotational speed is 2500rpm, is gone
Clear liquid, obtains the second sediment；
The 8ml buffer solutions that the second sediments of 16ml embodiment two is provided are resuspended, and obtain the second resuspended bacterium solution；
10min will be centrifuged under the conditions of 4 DEG C of second re-suspension liquid, and centrifugal rotational speed is 2500rpm, removes supernatant, obtains the 3rd
Sediment；
The sediments of 8ml the 3rd are placed in 3.72ml buffer solutions and the 0.28ml dimethyl Asia that the embodiment after precooling two is provided
It is resuspended in the mixed liquor of sulfone, after ice bath 20min, the temperature of the buffer solution of precooling and the mixed liquor of dimethyl sulfoxide (DMSO) is 4 DEG C,
3rd sediment of 8ml resuspended bacterium solutions formation, obtains competent cell.The competent cell prepared is stored in into -70 DEG C to treat
With.The competent cell of gained is used for plasmid conversion (identical with the plasmid of embodiment one), conversion process refers to Molecular
Cloning:A Laboratory Mannual Plays conversion process, obtains transformant 1050, transformation efficiency is 1.05 altogether
×109Cfu/ μ g, its experimental result is as shown in Figure 6.
Embodiment three
Be used to preparing the buffer solution of competent cell the embodiments of the invention provide a kind of, the buffer solution be include piperazine-
The ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- (2- ethoxys)-Tau, calcium chloride, the aqueous solution of potassium chloride and manganese chloride, the buffering
Liquid pH value is 7.0, in the present embodiment, and 9.0708g piperazines-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, 4.265g N, N- are added into buffer solution
(2- ethoxys)-Tau, 2.7745g calcium chloride, 22.3653g potassium chloride and 8.094g manganese chlorides, add water
1000ml so that the piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- (2- ethoxys)-Tau, calcium chloride, potassium chloride and chlorination
The concentration of manganese is respectively 30mM, 20mM, 25mM, 300mM and 50mM, when preparing buffer solution, and buffer solution is adjusted with potassium hydroxide
PH value, and filtration sterilization is stand-by in 4 DEG C by the buffer preserving prepared.
The recipient bacterium selected in the present embodiment is Escherichia coli, specially bacillus coli DH 5 alpha.
The bacterium solution of recipient bacterium is coated on plating medium, plating medium incubated overnight at 37 DEG C is inverted in, directly
Extremely occurs monoclonal on plating medium；
Picking monoclonal is placed in the shaking flask equipped with SOC Shake flask mediums, and 1L shaking flasks are equipped with 500ml SOC Shaking cultures
Base, shaken cultivation to OD600 values are 0.6 at 18 DEG C, and rotating speed during vibration is 100-110rpm；
SOC Shake flask mediums after culture are placed in 10min on ice, then by SOC Shaking cultures based on centrifugation under the conditions of 4 DEG C
10min, and centrifugal rotational speed is 2500rpm, removes supernatant, obtains the first sediment；
The 20ml buffer solutions that the first sediments of 500ml embodiment three is provided are resuspended, and obtain the first re-suspension liquid；
First re-suspension liquid ice bath 10min is centrifuged into 10min under the conditions of 4 DEG C, and centrifugal rotational speed is 2500rpm, is gone
Clear liquid, obtains the second sediment；
The 8ml buffer solutions that the second sediments of 24ml embodiment three is provided are resuspended, and obtain the second resuspended bacterium solution；
10min will be centrifuged under the conditions of 4 DEG C of second re-suspension liquid, and centrifugal rotational speed is 2500rpm, removes supernatant, obtain the 3rd
Sediment；
The sediments of 8ml the 3rd are placed in 3.72ml buffer solutions and the 0.28ml dimethyl Asia that the embodiment after precooling three is provided
It is resuspended in the mixed liquor of sulfone, after ice bath 25min, the temperature of the buffer solution of precooling and the mixed liquor of dimethyl sulfoxide (DMSO) is 4 DEG C,
Obtain competent cell.By the competent cell prepared be stored in -70 DEG C it is stand-by.The competent cell of gained is used for plasmid
Conversion (identical with the plasmid of embodiment one), conversion process refers to Molecular Cloning:A Laboratory Mannual
Plays conversion process, obtains transformant 2467 altogether, and transformation efficiency is 2.467 × 109Cfu/ μ g, its experimental result such as Fig. 7
It is shown.
Buffer solution provided in an embodiment of the present invention is used for when preparing chemical method competent cell, wherein calcium chloride, potassium chloride
Bacterial cell can be stimulated with the cation in manganese chloride, recipient bacterium is become the form of acceptant exogenous DNA, piperazine-
The ethyl sulfonic acid of Isosorbide-5-Nitrae-two can stablize cytoactive, and by adding N, N- (2- ethoxys)-Tau can ensure thin
On the premise of cytoactive, increase membrane passage, so that the transformation efficiency of the competent cell prepared is greatly improved, and
Gained competent cell can be applied to the conversion of long segment (the connection carrier that such as fragment length is 8100bp), increase cell membrane
Permeability after cause short-movie section be easier to be transferred in the competent cell；Meanwhile, preparation method provided in an embodiment of the present invention
Competent cell can efficiently and rapidly be prepared.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Sequence table
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<160> 2
<170>PatentIn version 3.4
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Claims (10)
1. a kind of buffer solution for being used to prepare competent cell, it is characterised in that the buffer solution is to include piperazine-Isosorbide-5-Nitrae-diethyl
Sulfonic acid, N, N- (2- ethoxys)-Tau, calcium chloride, the aqueous solution of potassium chloride and manganese chloride, the pH of the buffer solution
It is worth for 6.0~7.0, piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- (2- ethoxys)-Tau, chlorination in the buffer solution
The concentration of calcium, potassium chloride and manganese chloride is respectively 11~30mM, 3~20mM, 5~25mM, 50~300mM and 10~50mM.
2. buffer solution according to claim 1, it is characterised in that piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- in the buffer solution
(2- ethoxys)-Tau, calcium chloride, the concentration of potassium chloride and manganese chloride are respectively 12~20mM, 5~15mM, 10
~20mM, 150~250mM and 30~40mM, the pH value of the buffer solution is 6.7.
3. buffer solution according to claim 2, it is characterised in that piperazine-ethyl sulfonic acid of Isosorbide-5-Nitrae-two, N, N- in the buffer solution
(2- ethoxys)-Tau, calcium chloride, the concentration of potassium chloride and manganese chloride are respectively 15mM, 10mM, 15mM, 200mM
And 35mM.
4. a kind of method that competent cell is prepared using buffer solution as claimed in claim 1, it is characterised in that methods described
Including：
Monoclonal and the centrifugation of recipient bacterium are bred, the first sediment is obtained；
First sediment is resuspended with the buffer solution, the first resuspended bacterium solution is obtained；
It will be centrifuged after the first re-suspension liquid ice bath, remove supernatant, obtain the second sediment；
Second sediment is resuspended with the buffer solution, the second resuspended bacterium solution is obtained；
By second re-suspension liquid centrifugation, supernatant is removed, the 3rd sediment is obtained；
3rd sediment is placed in the mixed liquor of the buffer solution and dimethyl sulfoxide (DMSO) and is resuspended, ice bath 20~
After 30min, competent cell is obtained.
5. method according to claim 4, it is characterised in that the volume ratio of first sediment and the buffer solution
125:(4~8).
6. method according to claim 4, it is characterised in that the volume ratio of second sediment and the buffering is (2
~3):1.
7. method according to claim 4, it is characterised in that the buffering that the 3rd sediment is placed in after precooling
The mixed liquor of liquid and dimethyl sulfoxide (DMSO) is resuspended, the body of the 3rd sediment, the buffer solution and the dimethyl sulfoxide (DMSO)
Product is than being 200:93:7.
8. method according to claim 7, it is characterised in that the buffer solution and the mixed liquor of dimethyl sulfoxide (DMSO) of precooling
Temperature be 4 DEG C.
9. method according to claim 4, it is characterised in that the preparation method of the monoclonal of the recipient bacterium includes：Will
The bacterium solution of the recipient bacterium is coated on plating medium, and the plating medium is inverted at 37 DEG C and cultivated, until in institute
State and occur monoclonal on plating medium.
10. method according to claim 4, it is characterised in that the monoclonal of the breeding recipient bacterium simultaneously centrifuges bag
Include：Monoclonal described in picking is placed in SOC Shake flask mediums, and shaken cultivation is to OD at 15~20 DEG C600It is worth for 0.6；Will culture
The SOC Shake flask mediums afterwards are placed in after 10min on ice and centrifuged.
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