Preparation of escherichia coli competent cells and transformation method of escherichia coli competent cells

Abstract

The invention relates to preparation of escherichia coli competent cells and a transformation method of the escherichia coli competent cells. The preparation comprises following the steps of culturing the escherichia coli in a SOB liquid medium, adding a buffer solution I when OD 600 is 0.4 to 0.6, centrifugally collecting cells after ice bath, after a buffer solution II resuspends the cells, respectively putting into pre-cooling sterile centrifuge tubes to obtain the scherichia coli competent cells. In a transformation process, uniformly mixing DNA (deoxyribonucleic acid) with the competent cells, and directly coating the culture medium with the mixture after heat-shock, wherein ice bath and recovery steps are not needed. The escherichia coli competent cells are prepared by adopting buffer solutions of a special formula, the transformation efficiency can reach 1.23X109 cfu / mug pUC19 DNA. The method is simple in operation, high in transformation efficiency and good in repeatability, the preparation of the escherichia coli competent cells and DNA transforming operation are simplified, and a technological base is provided for high-efficiency and rapid transformation of the DNA.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to the preparation and transformation method of Escherichia coli competent cells. Background technique [0002] Escherichia coli can take up exogenous DNA (PlasmidDNA, PhageDNA, etc.) after being treated, and cells in this state are called "competent cells". The process of introducing foreign DNA molecules into recipient cells to make them acquire new genetic traits is called "transformation". In microbiology, molecular biology, genetic engineering and other research fields, it is often necessary to use various competent cells for DNA transformation operations. [0003] The preparation of competent cells is an important link in molecular biology research, and its quality directly affects the subsequent experimental work. Commonly used methods for preparing competent cells include CaCl 2 method, Hanahan and Inoue method. CaCl 2 Method (CohenSN, ChangACY, Hs...

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Problems solved by technology

CaCl 2 Method (CohenSN, ChangACY, HsuL.Nonchromosomalantibioticresistanceinbacteria:genetictransformationof Escherichiacoli byR-factorDNA.Proc.Natl.Acad.Sci.USA.1972,69:2110-2114.) is a conventional method for preparing competent cells in the laboratory. This method is simple and easy, but the transformation efficiency of the prepared competent cells is relatively low Low, can only meet the needs of routine molecular biology experiments; Hanahan method (HanahanD.Studies on transformation of Escherichiacoli withplasmid.J.Mol.Biol.1983,166:557-580.) The operator's skills, carefulness and reagent purity requirements are high, and the repeatability is low; Inoue method (InoueH., HNojimaandOkayamaH.Highefficiencytransformationof Escherichiacoli withplasmids.Gene.1990,96:23-28.) Although the transformation efficiency of competent cells is high, the OD of bacteria 600 The value requirements are relatively strict, and the cultivation temperature is 18°C. Bacteria grow slowly at this temperature, and the cultivation time is relatively long (about 20~50h), and the process is relatively cumbersome.

[0004] At present, the preparation and transformation process of competent cells is very cumbersome. The preparation process requires multiple centrifugation to collect cells and resuspend cells. time to culture recovery, greatly reducing experimental efficiency

In addition, the transformation efficiency of competent cells prepared by conventional methods is low, which cannot meet the requirements of special biological experiments (such as DNA library construction, transformation of large fragments of DNA)

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