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Preparation method for efficient competent cell of escherichia coli

A technology of competent cells and Escherichia coli, applied in the field of molecular biology, can solve the problems of reduced transformation efficiency, time-consuming and labor-intensive, application limitations, etc., and achieves the effects of high transformation efficiency, simple operation and long storage time.

Inactive Publication Date: 2013-04-17
康盈创新生物技术(北京)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are two ways to obtain E. coli competent cell stocks. One is to purchase frozen competent cells through commercial channels. The transformation efficiency is reliable and stable, but it requires a lot of money and its application is limited.
Another method is to make the competent cell stock in the laboratory, and the commonly used method is CaCl 2 method, by using pre-cooled CaCl 2 Treat the cultured Escherichia coli strain to make it enter the competent state for transformation, but the transformation efficiency of the prepared competent cells will decrease significantly after long-term frozen storage, and repeated preparation is required, which is time-consuming and labor-intensive

Method used

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  • Preparation method for efficient competent cell of escherichia coli

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Effect test

Embodiment 1

[0011] Embodiment 1: The method for preparing competent cells of the present invention and detection of transformation efficiency.

[0012] The materials and reagents used in this example are as follows: Materials: pUC18 plasmid and recipient bacteria DH5α were purchased from Beijing Tianenze Gene Technology Co., Ltd.

[0013] Reagent:

[0014] LB liquid medium: 10 grams of protein, 5 grams of yeast extract, 10 grams of sodium chloride, 2.4 grams of magnesium sulfate, distilled water to 1000ml, pH 7.0, sterilized at 121°C for 20 minutes.

[0015] The composition of TFB I solution is: 30mmol / L KAc, 100mmol / L RbCl, 10mmol / L CaCl 2 , 50mmol / L MnCl 2 , 15% glycerin.

[0016] The composition of TFB II solution is: 10mmol / L MOPS, 75mmol / L CaCl 2 , 10mmol / L RbCl, 15% glycerol.

[0017] Competent cell preparation process of the present invention is as follows:

[0018] Spread Escherichia coli DH5α preservation solution on LB plates without antibiotics, culture overnight at 37°C;...

Embodiment 2

[0021] Example 2 Calcium Chloride Method Competent Cell Preparation Method and Transformation Efficiency Detection

[0022] Calcium chloride method competent cell preparation process is as follows (as a comparison):

[0023] Spread Escherichia coli DH5α preservation solution on LB plates without antibiotics, culture overnight at 37°C; pick up monoclonal bacteria to 5ml LB medium and shake overnight at 37°C; inoculate into 100ml LB medium at a ratio of 1% (v / v) In medium, shake vigorously for 2-3 hours, and the OD value is about 0.6; 4°C, 4000r / min centrifuge for 10min to collect the bacteria, resuspend the bacteria with 40ml of ice-cooled 0.1mol / L calcium chloride solution, and incubate on ice for 30min; 4°C , Centrifuge at 4000r / min for 10min to collect the bacteria, resuspend the bacteria in 4ml of ice-cooled 0.1mol / L calcium chloride solution (containing 15% glycerol), ice-bath for 15-60min, pack in 100ul / tube, store at -80°C for later use .

[0024] The conversion effici...

Embodiment 3

[0030] Example 3 Conversion Influencing Factors

[0031] Competent cells were prepared by Escherichia coli DH5α, and the competent cells prepared by the two methods were compared at four time points of immediate use, frozen storage (-80°C) for 3 months, 6 months, and 9 months. The plasmid pUC18 was transformed into bacteria, and the clones were counted. The result is as follows:

[0032]

[0033] Conclusion: From the above, it can be seen that the competent cells prepared by this method have no obvious changes when they are stored for 9 months, while the competent cells prepared by the calcium chloride method, as time goes on, the number of clones of competent cells decreases .

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Abstract

The invention provides a preparation method for an efficient competent cell of escherichia coli. The step comprises the steps that escherichia coli is cultivated in a conventional liquid medium till an OD value is about 0.4, then cells are collected by centrifugation, and the competent cell is prepared by a rubidium chloride method.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a preparation method of high-efficiency competent cells of Escherichia coli. Background technique [0002] Transformation is the process of introducing exogenous DNA molecules into recipient bacteria to obtain new genetic traits. It is one of the most basic operating techniques in modern molecular biology. Transformation efficiency directly affects the progress and work efficiency of previous and subsequent experiments, and the state of competent cells is one of the most direct and key factors affecting transformation efficiency. At present, there are two ways to obtain Escherichia coli competent cell storage. One is to purchase frozen competent cells through commercial channels. The transformation efficiency is reliable and stable, but it requires a lot of money and its application is limited. Another method is to make the competent cell stock in the labor...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12R1/19
Inventor 不公告发明人
Owner 康盈创新生物技术(北京)有限公司
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