Rapid and Efficient Method to Make Competent Bacterial Cells for Genetic Transformation

Beenzu Siamalube; Emmanuel Ehinmitan; Maina Ngotho; Justus Onguso; Steven

doi:10.15379/ijmst.v11i1.3758

Author(s)

Beenzu Siamalube Department of Molecular Biology and Biotechnology, Pan African University Institute for Basic Sciences, Technology and Innovation, P.O. Box 62000-00200, Nairobi, Kenya

Emmanuel Ehinmitan Department of Molecular Biology and Biotechnology, Pan African University Institute for Basic Sciences, Technology and Innovation, P.O. Box 62000-00200, Nairobi, Kenya

Maina Ngotho Department of Animal Sciences, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000-00200, Nairobi, Kenya

Justus Onguso Institute for Biotechnology Research, Jomo Kenyatta University of Agriculture and Technology, P.O. Box 62000-00200, Nairobi, Kenya

Steven Department of Biochemistry, Microbiology and Biotechnology, Kenyatta University, P.O. Box 43844 GPO, 00100, Nairobi, Kenya

Abstract

There is absence of natural occurrence of genetic transformation in many bacteria due to the negative charges of DNA and bacteria, hence the DNA must be forced into the bacterial cells. Escherichia coli is mainly the host of choice because of its simplistic genetic mechanism and readily available molecular biological tools to inexpensively cultivate it. After the bacterial cells are made competent, it is anticipated that they contain and express the gene of interest that is cloned in the plasmid they take up during transformation. The Polymerase Chain Reaction method is then used to determine the presence of the gene and identify the band size, thereof. This paper will tabulate the step-by-step protocol of making competent E. coli cells strain K12 using calcium chloride solution and the heat-shock method and outline the procedure to confirm the positive transformants.

Publication Details

Page(s) 689-697

DOI 10.15379/ijmst.v11i1.3758

Published Journal: International Journal of Membrane Science and Technology, Volume: 11, Issue: 1, Year: 2024

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Keywords

Genetic transformation, coli, E, CaCl2 solution, competent cell, LB media, plasmid DNA

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