CN114672506A - Transformation method of escherichia coli - Google Patents
Transformation method of escherichia coli Download PDFInfo
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- CN114672506A CN114672506A CN202210277096.0A CN202210277096A CN114672506A CN 114672506 A CN114672506 A CN 114672506A CN 202210277096 A CN202210277096 A CN 202210277096A CN 114672506 A CN114672506 A CN 114672506A
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Abstract
The invention discloses a transformation method of escherichia coli, and relates to a transformation method of microbial strains. The invention solves the problems of longer reaction time and complicated process in the existing escherichia coli transformation technology. The method comprises the following steps: firstly, the plasmid and 30ul of escherichia coli are gently mixed and placed on ice, then heat shock is carried out in water bath at 42 ℃, then the plasmid is immediately placed back into the ice, and the mixture is kept stand for 1 min; finally, inoculating the culture medium into a solid LB plate culture dish containing antibiotics for culture. The method has short reaction time and simple process, and is beneficial to popularization and application.
Description
Technical Field
The invention relates to a method for transforming microbial strains.
Background
At present, the transformation technology has become a basic operation method of recombinant DNA technology, is widely applied to researches such as gene positioning, gene cloning, expression of exogenous genes, drawing of gene maps and the like, and is one of the main research technologies of molecular genetics. The principle of transformation is that the plasmid DNA is adhered to the surface of bacterial cell, and after short-time heat shock treatment, it promotes DNA absorption, and then the first generation is cultured in non-selective culture medium, and when the antibiotic gene carried on the plasmid is expressed, it can grow in the culture medium containing antibiotic. Escherichia coli (also known as Escherichia coli) is the most commonly used competent cell in gene cloning experiments due to its simple structure and clear genetic background, and can be applied to efficient DNA cloning and plasmid amplification, reduce the occurrence of homologous recombination of cloned DNA, and improve the yield and quality of plasmid DNA. The present transformation research is not only of theoretical significance, but also an important means for introducing plasmid or virus vector into host cell in genetic engineering (see recombinant DNA technology), and it may provide new approach for breeding and gene therapy of hereditary diseases. At present, transformation is mostly applied to vector construction, is widely applied in a cell level, and is an important early means for molecular mechanism research of a target gene in vitro. Currently, most optimization researches on the transformation technology of escherichia coli are carried out, and old eastern et al firstly activate and culture the escherichia coli overnight, and then directly coat the transformation mixture on the Ca-containing mixture precooled at 4 DEG C 2+The transformation was completed on solid LB plate petri dishes containing the antibiotic. The conventional 50m L centrifugal tube is changed into a 1.5m L EP tube by the substitute army, so that the pollution risk possibly generated in the subpackaging process is effectively reduced, the test procedure is simplified, and the conversion efficiency is improved. The heat stress at 42 ℃ is optimized to be placed for 10min at room temperature by Zhangzhu and the like, and the culture time of the mixture is shortened to beAnd (4) 40 min. Although the researches optimize the escherichia coli transformation technology to different degrees, the reaction time is long, an LB liquid culture medium is needed, and the process is complicated.
Disclosure of Invention
The invention provides a transformation method of escherichia coli, aiming at solving the problems of longer reaction time and complicated process in the existing escherichia coli transformation technology.
The transformation method of the escherichia coli is carried out according to the following steps: firstly, 10 mul of mixed liquid of the plasmid and 30ul of escherichia coli is placed on ice for 5min, then is placed in a water bath at 42 ℃ for 45s for heat shock, and then is immediately placed back in the ice and stands for 1 min; and then inoculating the mixed solution of the plasmid and the escherichia coli into a solid LB plate culture dish containing antibiotics, positively culturing for 1-2 h in a constant temperature environment of 37 ℃, and then inversely placing for 8-12 h to finish the transformation of the escherichia coli.
In the transformation process of the invention, the low temperature is firstly carried out after the Escherichia coli and the plasmid are mixed so as to lead the DNA-CaCl2The complex is adhered to the surface of the thallus, the permeability of the thallus wall is changed through transient heat shock, the thallus is kept stand on ice for 1min, and finally DNA attached to the surface enters the thallus, so that the transformation of the Escherichia coli is realized. In the process, the invention only needs 30ul of escherichia coli, and the escherichia coli can be transformed without shaking culture in an LB liquid culture medium in the whole transformation process. The method has short reaction time and simple process, and is beneficial to popularization and application.
Drawings
FIG. 1 is a photograph of the coated plate of example 1;
FIG. 2 shows the growth of colonies in the plastic plate of example 1 (optimized according to the invention);
FIG. 3 shows the growth of colonies in the glass plate of example 1 (optimization method of the present invention);
FIG. 4 is the colony growth in the plastic dish of example 2;
FIG. 5 shows the growth of colonies in the glass plate of example 2;
FIG. 6 is PCR electrophoresis chart of the bacterial liquid in example 1 and example 2;
FIG. 7 is the electrophoretogram after double cleavage in example 1 and example 2.
Detailed Description
The first embodiment is as follows: the method for transforming Escherichia coli according to the present embodiment is performed by the following steps: firstly, 10 mul of mixed liquid of the plasmid and 30ul of escherichia coli is placed on ice for 5min, then is placed in a water bath at 42 ℃ for 45s for heat shock, and then is immediately placed back in the ice and stands for 1 min; and then inoculating the mixed solution of the plasmid and the escherichia coli into a solid LB plate culture dish containing antibiotics, positively culturing for 1-2 h in a constant temperature environment of 37 ℃, and then inversely placing for 8-12 h to finish the transformation of the escherichia coli.
The plasmid DNA content described in this embodiment is no more than 100 ng.
The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: the plasmid is a plasmid containing a gene of interest. Other steps and parameters are the same as those in the first embodiment.
Example 1 transformation of E.coli Using the method of one of the embodiments
1. Preparation of test materials
1.1 apparatus
Vortex mixer, micropipette sampler, pipette tip, 1.5ml microcentrifuge tube, double-sided microcentrifuge tube rack, dry type constant temperature air bath (or constant temperature water bath kettle), ice maker, constant temperature shaking table, culture dish (paved solid LB-Amp: glass plate and disposable sterilized plastic plate), super clean bench, alcohol lamp, glass coating rod, constant temperature incubator.
1.2 reagents
Escherichia coli (competent taraka, 9057), plasmid DNA (plasmid into which the introduced target gene is ligated with PMD-18T), LB liquid medium (containing antibiotics).
2. Material treatment
Sterile 1.5ml centrifuge tubes, sterile 100ul and 10ul tips.
3. Transformation of E.coli
1) The temperature of the thermostatic water bath is adjusted to 42 ℃ in advance;
2) taking out a tube of competence (Escherichia coli) from a-80 deg.C ultra-low temperature freezer, and thawing on ice;
3) 10ul of plasmid DNA (wherein the DNA content does not exceed 100ng) was gently mixed with 30ul of E.coli and then placed on ice for 5 min.
4) After the ice bath is finished, performing heat shock in water bath at 42 ℃ for 45s, quickly putting back into ice, and standing for 1 min;
5) then, the mixed solution of the plasmid and the escherichia coli is uniformly coated on a solid LB plate culture dish containing antibiotics by using a glass coating rod burnt by an alcohol burner (the solid LB plate culture dish adopts a plastic plate and a glass plate respectively);
6) placing the solid LB plate culture medium containing the antibiotic inoculated with the mixed solution into a constant temperature incubator at 37 ℃, placing the resistance plate for 1-2h, and then placing the resistance plate upside down for 8-12h to finish the conversion; and closely observing the growth change of the colony;
7) randomly picking a single transformed escherichia coli colony from a solid LB plate culture medium containing antibiotics, putting the single transformed escherichia coli colony into a centrifugal tube of 1.5ml containing 700ul of a liquid culture medium, independently culturing on a constant-temperature shaking table at 37 ℃, performing PCR detection on a bacterial liquid after culturing for 4h, and performing agarose gel electrophoresis to determine the colony positive rate and the transformation efficiency;
8) and carrying out amplification culture on the positive bacterial liquid, carrying out double digestion experiment after the plasmid is slightly extracted, and determining whether the connection is correct by using agarose gel electrophoresis.
Example 2 transformation of E.coli Using the existing transformation method
1. Preparation of test materials
1.1 apparatus
Vortex mixer, micropipette sampler, pipette tip, 1.5ml microcentrifuge tube, double-sided microcentrifuge tube rack, dry type constant temperature air bath (or constant temperature water bath kettle), ice maker, constant temperature shaking table, culture dish (paved solid LB-Amp: glass plate and disposable sterilized plastic plate), super clean bench, alcohol lamp, glass coating rod, constant temperature incubator.
1.2 reagents
Coli (competent taraka, 9057), plasmid DNA (plasmid mixture after ligation of the gene with PMD-18T), LB liquid medium (with and without antibiotics).
2. Material treatment
Sterile 1.5ml centrifuge tubes, sterile 100ul and 10ul tips.
3. Transformation of E.coli
1) The temperature of the thermostatic water bath is adjusted to 42 ℃ in advance;
2) taking out a tube of competence (Escherichia coli) from a-80 deg.C ultra-low temperature freezer, and thawing on ice;
3) 10ul of ligated plasmid (DNA content not exceeding 100ng) was gently mixed with 100ul of E.coli and then placed on ice for 30 min.
4) After the ice bath is finished, performing heat shock in water bath at 42 ℃ for 45s, quickly putting back into ice, and standing for 3-5 min;
5) adding 500ul LB liquid culture medium (without antibiotic) into the mixture, mixing gently, shaking at 37 deg.C for 40min at 200 rpm;
6) Then, uniformly coating the mixed solution on a solid LB plate culture dish containing antibiotics by using a glass coating rod burnt by an alcohol burner (the solid LB plate culture dish adopts a plastic plate and a glass plate respectively);
7) putting a solid LB plate culture dish containing antibiotics into a constant-temperature incubator at 37 ℃, and putting a resistant plate in a positive way for 1-2h, and then putting the resistant plate in an inverted way for 8-12h to finish the conversion; during this period, the growth of colonies was closely observed;
8) randomly picking a single colony of transformed escherichia coli from an LB (lysogeny broth) plate culture medium, putting the single colony into a 1.5ml centrifugal tube containing 700ul of an LB liquid culture medium (containing antibiotics), performing independent culture on a constant-temperature shaking table at 37 ℃, performing PCR (polymerase chain reaction) detection on a bacterium solution after culturing for 4h, and performing agarose gel electrophoresis to determine the colony positive rate and the transformation efficiency;
9) and carrying out amplification culture on positive bacteria liquid, carrying out double digestion experiments after small plasmid extraction, and determining whether the connection is correct by using agarose gel electrophoresis.
In the embodiment, pictures after coating are shown in fig. 1, a represents a flat plate of the optimization method of the invention, and b represents a flat plate of the prior art; FIG. 2 shows the growth of colonies in plastic plates according to example 1 (optimization method of the present invention); FIG. 3 shows the growth of colonies in a glass plate according to example 1 (optimization method of the invention); FIG. 4 is the colony growth in the plastic plate of example 2 (prior art transformation); FIG. 5 is the colony growth in the glass plate of example 2 (prior art transformation); FIG. 6 is PCR electrophoretogram of bacterial suspension of example 1 and example 2, wherein a represents the bacterial suspension of the optimization method of the present invention; b represents a bacterial liquid of the conventional transformation method; FIG. 7 electrophoretogram after double digestion of example 1 and example 2, wherein a represents DNA of the optimization method of the present invention; b represents DNA of a conventional transformation method. As can be seen from fig. 1 to 7, the number of colonies: the method of the present invention produces a greater number of colonies, while the number of colonies of the prior art is relatively smaller.
By comparing example 1 with example 2, it can be seen that:
example 1 the low temperature after mixing of E.coli and plasmid during transformation was for DNA-CaCl2The complex is adhered to the surface of the thallus, the permeability of the thallus wall is changed through transient heat shock, the thallus is kept stand on ice for 1min, and finally DNA attached to the surface enters the thallus, so that the transformation of the Escherichia coli is realized. Therefore, the low temperature only needs to ensure DNA-CaCl2The complex can be adhered to the surface of the thallus, and the method of the invention can ensure DNA-CaCl only by low temperature of 5min2The complex can adhere to the surface of the thallus;
example 2 after the mixture of plasmid and Escherichia coli is kept still on ice after heat shock, 500ul of LB medium (without antibiotic) is added into the mixture, and 100-200 ul of the mixture is spread on a resistant plate after constant temperature shaking culture for 40 min; the transformation method directly coats the plasmid and the Escherichia coli mixture into a resistant plate after standing on ice after heat shock, and the combined concentration of 10ul of plasmid and 30ul of Escherichia coli after standing on ice after heat shock can meet the requirement of the strain for growth on the resistant plate.
The method has the advantages of short reaction time, conversion step saving and simple and convenient conversion process.
Claims (2)
1. The transformation method of the Escherichia coli is characterized by comprising the following steps of: firstly, 10 mul of mixed liquid of the plasmid and 30ul of escherichia coli is placed on ice for 5min, then is placed in water bath at 42 ℃ for 45s for heat shock, and then is immediately placed back into the ice and stands for 1 min; and then inoculating the mixed solution of the plasmid and the escherichia coli into a solid LB plate culture dish containing antibiotics, positively culturing for 1-2 h in a constant temperature environment of 37 ℃, and then inversely placing for 8-12 h to finish the transformation of the escherichia coli.
2. The method of claim 1, wherein the plasmid is a plasmid containing the gene of interest.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030138956A1 (en) * | 2001-11-09 | 2003-07-24 | Tzu-Chih Chen | Fast method of transforming competent cells |
| US20070212783A1 (en) * | 2006-03-08 | 2007-09-13 | Tzu-Chih Chen | Fast method of transforming competent cells |
| CN106755039A (en) * | 2016-12-20 | 2017-05-31 | 南京农业大学 | A kind of method for improving Bacterial Transformation efficiency |
-
2022
- 2022-03-21 CN CN202210277096.0A patent/CN114672506A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030138956A1 (en) * | 2001-11-09 | 2003-07-24 | Tzu-Chih Chen | Fast method of transforming competent cells |
| US20070212783A1 (en) * | 2006-03-08 | 2007-09-13 | Tzu-Chih Chen | Fast method of transforming competent cells |
| CN106755039A (en) * | 2016-12-20 | 2017-05-31 | 南京农业大学 | A kind of method for improving Bacterial Transformation efficiency |
Non-Patent Citations (4)
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| 刘卫今等: "简单高效的感受态细胞制备和质粒转化体系的建立", 《安徽农业科学》 * |
| 杜祯等: "工程菌DH_(5a)感受态细胞的制备及质粒pGEM的转化研究", 《中国畜牧兽医》 * |
| 郑玉忠等: "《分子生物学实验教学中DNA转化方法的简化》", 《时代教育》 * |
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