Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.
Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples.
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.|Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany.|Animal Nutrition and Human Health Laboratory, College of Life Science of Hunan Normal University, 410081 Changsha, People's Republic of China.
Qiang
Q
Tu
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.|Department of Genomics, Dresden University of Technology, BioInnovations-Zentrum, Tatzberg 47-51, 01307 Dresden, Germany.|Animal Nutrition and Human Health Laboratory, College of Life Science of Hunan Normal University, 410081 Changsha, People's Republic of China.
Jia
J
Yin
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.|Department of Genomics, Dresden University of Technology, BioInnovations-Zentrum, Tatzberg 47-51, 01307 Dresden, Germany.
Jun
J
Fu
Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany.
Jennifer
J
Herrmann
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.
Yuezhong
Y
Li
Animal Nutrition and Human Health Laboratory, College of Life Science of Hunan Normal University, 410081 Changsha, People's Republic of China.
Yulong
Y
Yin
Department of Genomics, Dresden University of Technology, BioInnovations-Zentrum, Tatzberg 47-51, 01307 Dresden, Germany.
A Francis
AF
Stewart
Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany.
Rolf
R
Müller
Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong University, Shanda Nanlu 27, 250100 Jinan, People's Republic of China.
Youming
Y
Zhang
D001419:Bacteria / Q000235:genetics* / Q000648:ultrastructure; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics / Q000648:ultrastructure; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D013696:Temperature; D014169:Transformation, Bacterial