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Buffer solution for preparing competent cells and method for preparing competent cells

A technology of competent cells and buffers, which is applied in the field of preparing competent cell buffers and preparing competent cells, can solve the problems of low transformation efficiency of competent cells, achieve efficient and rapid preparation, increase permeability, and transformation efficiency Improved effect

Inactive Publication Date: 2017-08-11
JIANGHAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem of low transformation efficiency of the preparation of competent cells by chemical methods in the prior art, the embodiment of the present invention provides a buffer for preparing competent cells and a method for preparing competent cells

Method used

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  • Buffer solution for preparing competent cells and method for preparing competent cells
  • Buffer solution for preparing competent cells and method for preparing competent cells
  • Buffer solution for preparing competent cells and method for preparing competent cells

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Embodiment 1

[0034] The embodiment of the present invention provides a buffer solution for preparing competent cells, the buffer solution includes piperazine-1,4-diethanesulfonic acid, N,N-(2-hydroxyethyl)-2-amino An aqueous solution of ethanesulfonic acid, calcium chloride, potassium chloride and manganese chloride, the pH of the buffer is 6.7, in this example, 4.5354 g of piperazine-1,4-diethylsulfonate is added to the buffer Acid, 2.1325g N,N-(2-hydroxyethyl)-2-aminoethanesulfonic acid, 1.6647g calcium chloride, 14.9102g potassium chloride and 5.6656g manganese chloride, add water 1000ml, make piperazine-1, The concentrations of 4-diethanesulfonic acid, N,N-(2-hydroxyethyl)-2-aminoethanesulfonic acid, calcium chloride, potassium chloride and manganese chloride were 15mM, 10mM, 15mM, 200mM and 35mM, respectively , when preparing the buffer solution, adjust the pH value of the buffer solution with potassium hydroxide, and filter and sterilize, store the prepared buffer solution at 4°C for...

Embodiment 2

[0058] The embodiment of the present invention provides a buffer solution for preparing competent cells, the buffer solution includes piperazine-1,4-diethanesulfonic acid, N,N-(2-hydroxyethyl)-2-amino Aqueous solution of ethanesulfonic acid, calcium chloride, potassium chloride and manganese chloride, the pH of the buffer is 6.0, in this example, 3.326 g of piperazine-1,4-diethylsulfonate was added to 1 L of buffer Acid, 0.6398g N,N-(2-hydroxyethyl)-2-aminoethanesulfonic acid, 0.5549g calcium chloride, 3.7276g potassium chloride and 1.6187g manganese chloride, add water 1000ml, make piperazine-1, The concentrations of 4-diethanesulfonic acid, N,N-(2-hydroxyethyl)-2-aminoethanesulfonic acid, calcium chloride, potassium chloride and manganese chloride were 11 mM, 3 mM, 5 mM, 50 mM and 10 mM, respectively . Store the prepared buffer at 4°C until use.

[0059] The recipient bacterium selected in this embodiment is Escherichia coli, specifically Escherichia coli DH5α.

[0060] S...

Embodiment 3

[0069] The embodiment of the present invention provides a buffer solution for preparing competent cells, the buffer solution includes piperazine-1,4-diethanesulfonic acid, N,N-(2-hydroxyethyl)-2-amino Aqueous solution of ethanesulfonic acid, calcium chloride, potassium chloride and manganese chloride, the pH of the buffer is 7.0, in this example, 9.0708 g of piperazine-1,4-diethanesulfonic acid was added to the buffer , 4.265g N,N-(2-hydroxyethyl)-2-aminoethanesulfonic acid, 2.7745g calcium chloride, 22.3653g potassium chloride and 8.094g manganese chloride, add water 1000ml, make piperazine-1,4 - The concentrations of diethanesulfonic acid, N,N-(2-hydroxyethyl)-2-aminoethanesulfonic acid, calcium chloride, potassium chloride and manganese chloride are 30mM, 20mM, 25mM, 300mM and 50mM, respectively, When preparing the buffer solution, adjust the pH value of the buffer solution with potassium hydroxide, filter and sterilize, and store the prepared buffer solution at 4° C. until...

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Abstract

The invention discloses a buffer solution for preparing competent cells and a method for preparing the competent cells, belonging to the field of biotechnologies. The buffer solution is an aqueous solution containing piperazine-1,4-bisethanesulfonic acid, N,N-(2-hydroxyethyl)-2-aminoethanesulfonic acid, calcium chloride, potassium chloride and manganese chloride, wherein the pH value of the buffer solution is 6.0-7.0, and the concentrations of piperazine-1,4-bisethanesulfonic acid, N,N-(2-hydroxyethyl)-2-aminoethanesulfonic acid, calcium chloride, potassium chloride and manganese chloride are respectively 5-30 mM, 3-20 mM, 5-25 mM, 50-300 mM and 10-50 mM. For the buffer solution provided by the embodiment of the invention, by adding N,N-(2-hydroxyethyl)-2-aminoethanesulfonic acid, the permeability of the cell membrane can be increased, thus the conversion efficiency of the prepared competent cells is greatly improved, meanwhile, the prepared competent cells are suitable for transformation of long fragments, and moreover, with the preparation method provided by the embodiment of the invention, the competent cells can be efficiently and rapidly prepared.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a buffer for preparing competent cells and a method for preparing competent cells. Background technique [0002] The method of transforming recipient bacteria with exogenous DNA is one of the most commonly used methods in molecular biology research, and exogenous DNA includes plasmids and linking vectors. Exogenous DNA must be transferred into appropriate recipient bacteria for propagation in order to obtain a large number of positive recombinant clones or express the target gene product. [0003] The existing method is to culture the recipient bacteria in LB plate medium, pick a single colony and inoculate it in SOB (SuperOptimal Broth) shake flask medium for fermentation to realize the reproduction of a single colony, and shake culture at 18°C ​​to OD 600 Value is 0.6, ice bath for 10min, centrifuge at 4°C for 10min, bacteria suspended in pre-cooled TB buffer, ice-bathed for 10min...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12R1/19
CPCC12N1/20C12N1/005
Inventor 郑瑜刘晶晶常文文
Owner JIANGHAN UNIVERSITY
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