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Method for preparing novel competent cell and transformation step thereof

A competent cell, a new type of technology, applied in the field of molecular biology, can solve the problems of decreased sensory effect, reduced experimental efficiency, cumbersome transformation process, etc., and achieves the effect of increasing permeability, high application value, and good repeatability.

Active Publication Date: 2014-05-21
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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  • Method for preparing novel competent cell and transformation step thereof
  • Method for preparing novel competent cell and transformation step thereof
  • Method for preparing novel competent cell and transformation step thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Detection of the conversion time and conversion efficiency of the C-competent cell described in 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-compe...

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 / L CaCl 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 an antibiotic-free LB plate, and 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 vigo...

Embodiment 3

[0045] Embodiment 3 conversion influence factors

[0046] Comparing the C-competent cell described in the present invention and ordinary competent cells in the four time points of immediate use, frozen storage (-80°C) for 3 months, frozen storage for 6 months, and frozen storage for 9 months, the same plasmid pUC18 was used to carry out bacterial For the difference in transformation efficiency, the C-competent cells and ordinary competent cells at the above four time points were selected 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 they are stored for 9 months, while the number of clones of ordinary 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 Applications(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
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