CN106047753A - Preparation method of competent cells with high transformation efficiency - Google Patents

Preparation method of competent cells with high transformation efficiency Download PDF

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CN106047753A
CN106047753A CN201610397054.5A CN201610397054A CN106047753A CN 106047753 A CN106047753 A CN 106047753A CN 201610397054 A CN201610397054 A CN 201610397054A CN 106047753 A CN106047753 A CN 106047753A
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preparation
transformation efficiency
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CN106047753B (en
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万浩强
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Xiamen Life Interconnect Technology Co Ltd
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

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Abstract

The invention discloses a preparation method of competent cells with high transformation efficiency, relates to the technical field of microorganisms and solves the problems of inconvenience in operation, low transformation efficiency and low yield in the prior art. The preparation method comprises steps as follows: (1) strains are scribed on an LB panel and cultured overnight at the temperature of 37 DEG C; (2) an SOC culture medium is inoculated with the strains, and the strains are subjected to shake culture at the temperature of 18 DEG C under the condition of 250 rpm until the OD600 value ranges from 2.0 to 5.0; (3) a strain solution is centrifuged in a sterilized tube with the volume being 50 mL at the temperature of 4 DEG C under the condition of 2,500 g/5 min, a supernatant is discarded, a buffer solution is added to settled strains, and manual resuspension is performed; (4) a product is centrifuged at the temperature of 4 DEG C under the condition of 2,500 g/5 min, a supernatant is discarded, a proper quantity of buffer solutions and a proper amount of DMSO are added, the OD600 value is 5.0, and the final concentration of DMSO is 7%; (5) the product is subpackaged in a sterile EP tube, put in liquid nitrogen and frozen quickly, and the competent cells are obtained. The preparation method is simple to operate, the yield is high, and the transformation efficiency can be up to (1*10<8>)-(5*10<9>).

Description

A kind of preparation method of high transformation efficiency competent cell
Technical field
The present invention relates to microbial technology field, be specifically related to the preparation method of a kind of high transformation efficiency competent cell.
Technical background
The process entering host cell with the DNA molecular of hereditary information is made to be referred to as " conversion "；Through physically or chemically side The process of formula and there is the host cell that acceptant additional DNA segments enters, be referred to as " competent cell ".At prokaryote In, whether conversion is a more universal phenomenon, convert at iuntercellular and occur, on the one hand depend on donor bacterium and recipient bacterium Whether sibship during evolution, be on the other hand also in a kind of impression state and have a very large relationship with recipient bacterium. The state of competent cell directly affects the efficiency of conversion, in order to make host cell carry out specific DNA or the duplication of protein or Express, it is thus achieved that the competent cell being prone to convert is crucial.
Escherichia coli have growth and quickly, easily cultivate the advantages such as more thorough with research, have the most become all biological skills Art related experiment is most widely used as the microorganism of host cell.The method preparing competent cell mainly includes physical method Class big with chemical method two.The most more common physical method includes electricity irritation, ultrasound wave, osmotic pressure etc.；Chemical method includes CaCl2, RbCl (KCl), Inoue method etc..Chemical method has quick, stable, reproducible, the advantage that bacterial strain is applied widely, But there is the shortcoming that operation inconvenience, conversion ratio and yield are low in existing chemical method.
In order to solve the drawbacks described above that prior art exists, the present invention have developed that a kind of transformation efficiency is high, simple to operate, becomes The preparation method of this cheap competent cell so that competent cell transformation efficiency is up to 1 × 108-5×109。
Summary of the invention
The present invention is the problem low in order to solve operation inconvenience, conversion ratio and the yield of prior art existence, and provides A kind of preparation method of high transformation efficiency competent cell.
The present invention is achieved by the following technical solutions:
A kind of preparation method of high transformation efficiency competent cell, step is:
(1) escherichia coli are obtained microorganism list bacterium colony in the flat lining out of the LB without resistance, 37 DEG C of incubated overnight；
(2) take microorganism list colony inoculation to cultivate in the 1L conical flask containing 250mL SOC culture medium, 18 DEG C, Under the conditions of 250rpm, concussion is cultivated to OD600Value is between 2.0-5.0；
(3) by concussion cultivate gained bacterium solution be transferred in sterilized 50mL pipe, 2500g/5min, under the conditions of 4 DEG C from The heart, abandons supernatant, is added by precipitation strain and shifts to an earlier date the buffer 4 DEG C of pre-coolings, the most resuspended；
(4) by the culture fluid of step (3) gained at 2500g/5min, centrifugal under the conditions of 4 DEG C, abandon supernatant, add buffer And DMSO, make resuspended rear OD600Value is 5.0, DMSO final concentration of 7%；
(5) culture fluid of step (4) gained is dispensed into EP pipe, is then placed in quick-freezing in liquid nitrogen, is stored in-80 DEG C of refrigerators In, to obtain final product.
Further, the preparation process of described SOC culture medium is: by 20g peptone, 5g yeast extract, 0.5g NaCl, 0.186g KCl, adds in 900mL ultra-pure water, adjusts PH to 7.0 with 1M HCl after being sufficiently stirred for dissolving, fixed with ultra-pure water Hold to 1L, after autoclave sterilization, add the 2M MgCl of 5mL sterilizing2, add the 1MGlucose of 20mL filtration sterilization, mixing Uniformly, 4 DEG C of preservations.
The preparation method of described buffer is: PIPES 10mM, CaCl215mM, KCl250mM, adjust with 0.5M KOH PH to 6.7, adds MnCl255mM, degerming with 0.22 μm membrane filtration after fully dissolving, load in the most sterilized bottle in 4 DEG C store.
Escherichia coli described in step (1) preserve in-80 DEG C of refrigerators.
The present invention selects nutritious SOC culture medium culturing escherichia coli, wherein glucose, KCl, MgCl2, Glucose, tryptone content is higher so that thalli growth state is more preferable；And the Low-temperature culture of 18 DEG C, relative to 37 DEG C, 18 Under DEG C cryogenic conditions, thalli growth is the slowest, it is easier to control the time needed for thalli growth, more difficult grows because of bacterium solution The too fast reduction making excessive concentration and cause transformation efficiency, so that the growth conditions of thalline is more controllable, transformation efficiency is more High；The OD of culture600Value is between 2.0-5.0, relative to traditional OD600It is worth between 0.3-0.5, OD of the present invention600Be worth across Degree is bigger, operation is easier to make bacterium solution growth concentration to reach required scope, and the yield of competent cell is higher, and operation is more Add simple；Ca2+The competent cell processed, its transformation efficiency typically can reach 5 × 106-2×107Transformant/plasmid DNA, this Invention buffer is at Ca2+On the basis of, combine bivalent metal ion Mn2+, DMSO material process antibacterial, then can make transformation efficiency Improve 100-1000 times, reach 1 × 108-5×109；By culture liquid nitrogen flash freezer, competent cell transformation efficiency can be avoided Decline.
Compared with prior art, the present invention is simple to operate, and competent cell yield is high, and transformation efficiency is up to 1 × 108-5× 109。
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, to the present invention It is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not used to Limit the present invention.
Embodiment 1
(1) the e. coli tg1 strain being saved in-80 DEG C on a small quantity is taken in the flat lining out of LB, 37 DEG C of incubated overnight；
(2) picking monoclonal is cultivated to the 1L conical flask containing 250mL SOC culture medium, trains under the conditions of 18 DEG C of 250rpm Support to OD600Value is between 2.0-3.0；Wherein cell density with the cell number in every milliliter of culture fluid 5 × 107An individual left side The right side is preferred, i.e. application logarithmic (log) phase or the antibacterial of logarithmic growth early stage, can be by measuring the OD of culture fluid600Value controls；
The preparation process of SOC culture medium is: by 20g peptone, 5g yeast extract, 0.5g NaCl, 0.186g KCl, Adding in 900mL ultra-pure water, adjust PH to 7.0 with 1M HCl, be settled to 1L with ultra-pure water after being sufficiently stirred for dissolving, High Temperature High Pressure is gone out After bacterium, add the 2M MgCl of 5mL sterilizing2, add the 1M Glucose of 20mL filtration sterilization, mix homogeneously, 4 DEG C of preservations；
(3) culture is transferred in sterilized 50mL pipe, centrifugal receipts bacterium under the conditions of 2500g/5min 4 DEG C；
(4) abandoning supernatant, add in advance at the buffer of 4 DEG C of pre-coolings, the most resuspended, this step can not acutely be rocked；
The preparation method of buffer is: by PIPES 10mM, CaCl215mM, KCl250mM, adjust PH extremely with 0.5M KOH 6.7, add MnCl255mM, 0.22 μm membrane filtration sterilizing, load in the most sterilized bottle in 4 DEG C of storages；
(5) centrifugal under the conditions of 2500g/5min 4 DEG C, abandon supernatant；Add appropriate amount of buffer solution and DMSO, make resuspended rear OD600 Value is 5.0, DMSO final concentration of 7%；
(6) subpackage 100 μ l often manages, and is then placed in quick-freezing in liquid nitrogen, it is ensured that liquid nitrogen did not had all EP to manage, and outwells institute in ice chest After having liquid nitrogen, place 10s (explosion-proof), then be stored in-80 DEG C of refrigerators, to obtain final product；
(7) used time takes out in thawed on ice, adds converted product and softly mixes, after placing 30min on ice, and 42 DEG C of thermal shocks 45s, coated plate.
Embodiment 2
(1) a small amount of strain is taken in the flat lining out of the LB without resistance, 37 DEG C of incubated overnight；
(2) picking monoclonal is cultivated to the 1L conical flask containing 250mL SOC culture medium, trains under the conditions of 18 DEG C of 250rpm Support to OD600Value is between 3.0-4.0；
The preparation process of SOC culture medium is: by 20g peptone, 5g yeast extract, 0.5g NaCl, 0.186g KCl, Adding in 900mL ultra-pure water, adjust PH to 7.0 with 1M HCl, be settled to 1L with ultra-pure water after being sufficiently stirred for dissolving, High Temperature High Pressure is gone out After bacterium, add the 2M MgCl of 5mL sterilizing2, add the 1M Glucose of 20mL filtration sterilization, mix homogeneously, 4 DEG C of preservations；
(3) culture is transferred in sterilized 50mL pipe, centrifugal receipts bacterium under the conditions of 2500g/5min 4 DEG C；
(4) abandoning supernatant, add in advance at the buffer of 4 DEG C of pre-coolings, the most resuspended, this step can not acutely be rocked；
The preparation method of buffer is: by PIPES 10mM, CaCl215mM, KCl250mM, adjust PH extremely with 0.5M KOH 6.7, add MnCl255mM, 0.22 μm membrane filtration sterilizing, load in the most sterilized bottle in 4 DEG C of storages；
(5) centrifugal under the conditions of 2500g/5min 4 DEG C, abandon supernatant；Add appropriate amount of buffer solution and DMSO, make resuspended rear OD600 Value is 5.0, DMSO final concentration of 7%；
(6) subpackage 100 μ l often manages, and is then placed in quick-freezing in liquid nitrogen, it is ensured that liquid nitrogen did not had all EP to manage, and outwells institute in ice chest After having liquid nitrogen, place 10s (explosion-proof), then be stored in-80 DEG C of refrigerators, to obtain final product；
(7) used time takes out in thawed on ice, adds converted product and softly mixes, after placing 30min on ice, and 42 DEG C of thermal shocks 45s, coated plate.
Embodiment 3
(1) a small amount of strain is taken in the flat lining out of the LB without resistance, 37 DEG C of incubated overnight；
(2) picking monoclonal is cultivated to the 1L conical flask containing 250mL SOC culture medium, trains under the conditions of 18 DEG C of 250rpm Support to OD600Value is between 4.0-5.0；
(3) preparation process of SOC culture medium is: by 20g peptone, 5g yeast extract, 0.5g NaCl, 0.186g KCl, adds in 900mL ultra-pure water, adjusts PH to 7.0 with 1M HCl, be settled to 1L with ultra-pure water after being sufficiently stirred for dissolving, and high temperature is high After pressure sterilizing, add the 2M MgCl of 5mL sterilizing2, add the 1M Glucose of 20mL filtration sterilization, mix homogeneously, 4 DEG C of preservations；
Culture is transferred in sterilized 50mL pipe, centrifugal receipts bacterium under the conditions of 2500g/5min 4 DEG C；
(4) abandoning supernatant, add in advance at the buffer of 4 DEG C of pre-coolings, the most resuspended, this step can not acutely be rocked；
The preparation method of buffer is: by PIPES 10mM, CaCl215mM, KCl250mM, adjust PH extremely with 0.5M KOH 6.7, add MnCl255mM, 0.22 μm membrane filtration sterilizing, load in the most sterilized bottle in 4 DEG C of storages；
(5) centrifugal under the conditions of 2500g/5min 4 DEG C, abandon supernatant；Add appropriate amount of buffer solution and DMSO, make resuspended rear OD600 Value is 5.0, DMSO final concentration of 7%；
(6) subpackage 100 μ l often manages, and is then placed in quick-freezing in liquid nitrogen, it is ensured that liquid nitrogen did not had all EP to manage, and outwells institute in ice chest After having liquid nitrogen, place 10s (explosion-proof), then be stored in-80 DEG C of refrigerators, to obtain final product；
(7) used time takes out in thawed on ice, adds converted product and softly mixes, after placing 30min on ice, and 42 DEG C of thermal shocks 45s, coated plate.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention is not limited thereto, Any those familiar with the art in the technical scope that the invention discloses, the change that can readily occur in or replacement, All should contain within protection scope of the present invention.Therefore, protection scope of the present invention should be with scope of the claims It is as the criterion.

Claims (3)

1. a preparation method for high transformation efficiency competent cell, the steps include:
(1) escherichia coli are obtained microorganism list bacterium colony in the flat lining out of the LB without resistance, 37 DEG C of incubated overnight；
(2) take microorganism list colony inoculation to cultivate in the 1L conical flask containing 250mL SOC culture medium, 18 DEG C, 250rpm bar Under part, concussion is cultivated to OD600Value is between 2.0-5.0；
(3) bacterium solution that concussion is cultivated gained is transferred in sterilized 50mL pipe, and 2500g/5min is centrifugal under the conditions of 4 DEG C, abandons Supernatant, adds precipitation strain and shifts to an earlier date the buffer 4 DEG C of pre-coolings, the most resuspended；
(4) by the culture fluid of step (3) gained at 2500g/5min, centrifugal under the conditions of 4 DEG C, abandon supernatant, add buffer and DMSO, makes resuspended rear OD600Value is 5.0, DMSO final concentration of 7%；
(5) culture fluid of step (4) gained is dispensed into EP pipe, is then placed in quick-freezing in liquid nitrogen, is stored in-80 DEG C of refrigerators, Obtain.
The preparation method of high transformation efficiency competent cell the most according to claim 1, it is characterised in that described SOC The preparation process of culture medium is: by 20g peptone, 5g yeast extract, 0.5g NaCl, 0.186g KCl, adds 900mL and surpasses In pure water, adjust PH to 7.0 with 1M HCl after being sufficiently stirred for dissolving, be settled to 1L with ultra-pure water, after autoclave sterilization, add The 2M MgCl of 5mL sterilizing2, add the 1M Glucose of 20mL filtration sterilization, mix homogeneously, 4 DEG C of preservations.
The preparation method of high transformation efficiency competent cell the most according to claim 1 and 2, it is characterised in that described The preparation method of buffer is: PIPES 10mM, CaCl215mM, KCl 250mM, adjusts PH to 6.7 with 0.5M KOH, adds MnCl255mM, degerming with 0.22 μm membrane filtration after fully dissolving, load in the most sterilized bottle in 4 DEG C of storages.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109852559A (en) * 2019-01-08 2019-06-07 海南医学院 A kind of preparation method of easy, efficient competent escherichia coli cell
CN110004078A (en) * 2019-03-20 2019-07-12 华南农业大学 A method of preparing high conversion Gram-negative clinic bacterium competence cell
CN113481229A (en) * 2021-07-08 2021-10-08 上海佐润生物科技有限公司 Preparation method for obtaining escherichia coli super competent cells
CN114574367A (en) * 2022-04-25 2022-06-03 济宁学院 Freeze-drying protective agent for escherichia coli DH5 alpha competent cells and use method and application thereof

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109852559A (en) * 2019-01-08 2019-06-07 海南医学院 A kind of preparation method of easy, efficient competent escherichia coli cell
CN110004078A (en) * 2019-03-20 2019-07-12 华南农业大学 A method of preparing high conversion Gram-negative clinic bacterium competence cell
CN110004078B (en) * 2019-03-20 2022-05-17 华南农业大学 Method for preparing high-conversion-rate gram-negative clinical bacteria competent cells
CN113481229A (en) * 2021-07-08 2021-10-08 上海佐润生物科技有限公司 Preparation method for obtaining escherichia coli super competent cells
CN114574367A (en) * 2022-04-25 2022-06-03 济宁学院 Freeze-drying protective agent for escherichia coli DH5 alpha competent cells and use method and application thereof

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