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A colony-to-lawn method for efficient transformation of Escherichia coli

Author:
An, Y., Lv, A., Wu, W.
Source:
Letters in applied microbiology 2010 v.51 no.1 pp. 98-103
ISSN:
0266-8254
Subject:
Escherichia coli, calcium chloride, clones, ethanol, freeze-thaw cycles, mutants, plasmids, protein synthesis, screening
Abstract:
To develop a fast, convenient, inexpensive and efficient Escherichia coli transformation method for changing hosts of plasmids, which can also facilitate the selection of positive clones after DNA ligation and transformation. A single fresh colony from plasmid-containing donor strain is picked up and suspended in 75% ethanol. Cells are pelleted and resuspended in CaCl₂ solution and lysed by repetitive freeze-thaw cycles to obtain plasmid-containing cell lysate. The E. coli recipient cells are scraped from the lawn of LB plate and directly suspended in the plasmid-containing cell lysate for transformation. Additionally, a process based on colony-to-lawn transformation and protein expression was designed and conveniently used to screen positive clones after DNA ligation and transformation. With this method, a single colony from plasmid-containing donor strain can be directly used to transform recipient cells scraped from lawn of LB plate. Additionally, in combination with this method, screening of positive clones after DNA ligation and transformation can be convenient and time-saving. Compared with current methods, this procedure saves the steps of plasmid extraction and competent cell preparation. Therefore, the method should be highly valuable especially for high-throughput changing hosts of plasmids during mutant library creation.
Agid:
2257347