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Preparation method and transformation steps of a novel competent cell

A technology of competent cells and bacteria, applied in the field of molecular biology, can solve the problems of cumbersome transformation process, decreased sensing effect, and reduced experimental efficiency, etc., and achieves high application value, increased permeability, and good repeatability.

Active Publication Date: 2016-01-13
简石生物技术(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most important thing for the efficiency of competent cells is to refrigerate. The freshly prepared competent cells have the best sensory effect, but the sensory effect will gradually decrease as time goes on.
Moreover, the transformation process is very cumbersome, requiring heat shock reactions, long-term cultivation, and a series of growth steps, which greatly reduce the experimental efficiency.

Method used

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  • Preparation method and transformation steps of a novel competent cell
  • Preparation method and transformation steps of a novel competent cell
  • Preparation method and transformation steps of a novel competent cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Detection of conversion time and conversion efficiency of C-competentcell according to the present invention.

[0022] Materials and reagents used in this embodiment are as follows:

[0023] Materials: pUC18 plasmid, recipient strain DH5α

[0024] Reagents: 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.

[0025] Buffer washing solution 1 is an aqueous solution containing the following components: 50 mM magnesium chloride, 20 mM potassium chloride, 100 mM calcium chloride, 1% glucose, 20 mM 4-hydroxyethylpiperazineethanesulfonic acid, and the pH is adjusted to pH 6.5.

[0026] Buffer washing solution 2 is an aqueous solution comprising the following components: 30 mM calcium chloride, 50 mM sodium chloride, 30 mM ferric chloride, 10% DMSO, adjusted to pH 7.0.

[0027] The preparation process of C-competentcell ...

Embodiment 2

[0034] Example 2 Common Competent Cell Preparation Method Transformation and Transformation Efficiency Detection

[0035] Materials and reagents used in this embodiment are as follows:

[0036] Materials: pUC18 plasmid, recipient strain DH5α

[0037] Reagents: 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, 0.1mol / LCaCl 2 (contains 15% glycerin)

[0038] The preparation process of ordinary competent cells (prepared by calcium chloride method) is as follows (for the steps, refer to the Molecular Biology Experiment Manual, as a comparison of C-competent cells):

[0039] Spread Escherichia coli DH5α preservation solution on LB plates without antibiotics, culture overnight at 37°C; pick up monoclonal bacteria and shake them in 5ml LB medium at 37°C overnight; inoculate them into 100ml LB medium at a ratio of 1% (v / v), Shake vigorou...

Embodiment 3

[0045] Embodiment 3 conversion influence factors

[0046] Comparing the C-competentcell described in the present invention and ordinary competent cells, using the same plasmid pUC18 for bacterial transformation at four time points of immediate use, frozen storage (-80°C) for 3 months, frozen storage for 6 months, and frozen storage for 9 months For the difference in efficiency, the C-competent cells and ordinary competent cells at the above four time points were taken for transformation, and the clones were counted after successful transformation.

[0047] The result is as follows:

[0048]

[0049] Conclusion: From the above, it can be seen that the C-competent cells described in the present invention have no obvious changes when stored for 9 months, while the number of clones of the common competent cells decreases with the prolongation of time.

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Abstract

The invention provides a method for preparing a novel competent cell (C-competent cell for short) and a transformation step thereof. The method comprises the following preparation processes: cultivating escherichia coli, centrifugally collecting bacteria, collecting the bacteria after re-suspending the bacteria by a buffer solution 1, collecting the bacteria after re-suspending the bacteria by a buffer solution 2; and split charging for later use. In the preparation method, the buffer solution 1 is a water solution containing the following components: magnesium chloride (10mM-1M), potassium chloride (10mM-1M), calcium chloride (10mM-1M), glucose (0.1-5%), and 4-hydroxyethylpiperazine ethane sulfonic acid (10mM-100mM), the pH value is 6.5; the buffer solution 2 is a water solution containing the following components: calcium chloride (10mM-1M), sodium chloride (10mM-1M), ferric chloride (10mM-1M), and dimethylsulfoxide (DMSO) (5-30%), and the pH value is 7.0. The transformation process comprises the following steps: thawing the C-competent cell on ice; adding to-be-transformed plasmid DNA when thawing for 1 / 3 hours; and evenly mixing and transforming into a cloning coated plate to cultivate.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a preparation method of a novel competent cell and a transformation step thereof. Background technique [0002] Competent cells refer to cells that are easy to accept foreign DNA after appropriate treatment, and foreign DNA molecules can be introduced into them to obtain new genetic traits. The transformation efficiency of competent cells directly affects the progress and work efficiency of experiments before and after, and the state of competent cells is one of the most direct and key factors affecting transformation efficiency. At present, the most important thing for the efficiency of competent cells is to refrigerate. The freshly prepared competent cells have the best sensing effect, but the sensing effect will gradually decrease as time goes on. Moreover, the transformation process is very cumbersome, requiring heat shock reaction, long-term cultivatio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/70C12R1/19
Inventor 不公告发明人
Owner 简石生物技术(北京)有限公司