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Plasmid production and purification: An integrated experiment‐based biochemistry and biotechnology laboratory course
Author(s) -
Santos Tiago,
Pereira Patrícia,
Queiroz João A.,
Cruz Carla,
Sousa Fani
Publication year - 2019
Publication title -
biochemistry and molecular biology education
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.34
H-Index - 39
eISSN - 1539-3429
pISSN - 1470-8175
DOI - 10.1002/bmb.21290
Subject(s) - plasmid , escherichia coli , lysis , biology , recombinant dna , lac operon , alkaline lysis , microbiology and biotechnology , agarose , biochemistry , chromatography , chemistry , dna , gene , dna vaccination
This laboratory experiment describes the production and purification of plasmid DNA for undergraduate biochemistry and biotechnology courses. This experiment performed in a one‐week period includes the protocols for plasmid pVAX1‐ LacZ production in Escherichia coli DH5 α cells and subsequent purification of supercoiled pVAX1‐ LacZ . Firstly, the students use a growth medium that favors the replication of the plasmid resulting in a higher plasmid production during exponential growth. Afterwards, alkaline lysis is done to disrupt the bacterial cells and recover pVAX1‐ LacZ plasmid. Lastly, they perform the purification of pVAX1‐ LacZ supercoiled isoform by L‐histidine chromatography, followed by agarose gel electrophoresis to characterize the separation of supercoiled isoform from contaminants. The proposed experiment provides an opportunity for students to acquire these skills that are routinely used in biochemistry and biotechnology laboratories. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):638–643, 2019.