Preparing method for electrically transforming competent cells without buffer solution

Abstract

The invention provides a preparing method for electrically transforming competent cells without a buffer solution, and belongs to the technical field of molekulare bakteriengenetik. The preparing method comprises the following steps that colibacillus is inoculated into a liquid culture medium containing magnesium ions for first culture; the first culture is mixed with the liquid culture medium, and then secondary culture is conducted to obtain a second culture, the second culture is subjected to centrifugal separation, and precipitation is obtained; the precipitation is mixed with water, afterthe precipitation is suspended, centrifugal separation is conducted, and precipitation is obtained; the precipitation is mixed with water, after the precipitation is suspended, centrifugal separationis conducted, precipitation is obtained, the operation is repeated one time, and precipitation is obtained; the precipitation is mixed with water, after the precipitation is suspended, centrifugal separation is conducted, and precipitation is obtained; the precipitation is mixed with water, after being suspended, the precipitation is evenly mixed with dimethyl sulfoxide, and electrically-transformed competent cells are obtained. By means of the preparing method, the high- transformation-rate competent cells can be obtained, the operation is simple, and the preparation cost is low.

Description

technical field [0001] The invention belongs to the technical field of bacterial molecular genetics, and in particular relates to a preparation method of electrotransformation competent cells without buffer solution. Background technique [0002] Escherichia coli can absorb exogenous DNA after being treated. Cells in this state are called "competent cells". The preparation of competent cells is an important link in molecular biology research, and the transformation efficiency directly affects the subsequent Experimental work carried out. Commonly used methods for preparing competent cells include CaCl 2 method, Hanahan and Inoue method. At present, the competent cells prepared by conventional methods require specific buffers in the preparation process, such as conventionally selected buffer components: the TB conversion buffer of every 20mL is composed of the following components: 12.5mL of double distilled water, 4mL of 1mol / L of KCl, 2.4mL of 0.45mol / L MnCl 2 , 0.5mol...

AI Technical Summary

Problems solved by technology

At present, for the competent cells prepared by conventional methods, the preparation process requires a specific buffer, such as the conventionally selected buffer composition: the TB transformation buffer of every 20mL is composed of the following components: 12.5mL of double distilled water, 4mL of 1mol / L of KCl, 2.4mL of 0.45mol / L MnCl 2 , 0.5mol / L of CaCl 2 0.6mL and 0.5mL of 0.5mol / L K-MES buffer, the buffer is not only cumbersome to prepare, but also expensive