Plasmid DNA Isolation and Characterization

Plasmid DNA was isolated from Listeria spp. and B. subtilis strains, as described previously [23 (link)]. In the case of S. aureus strains, lysostaphin (A&A Biotechnology, Gdańsk, Poland) was used instead of lysozyme (Sigma-Aldrich). Plasmid DNA for sequencing was isolated using large-scale preparation methods [80 (link)], and sequence assembly was verified by RFLP analysis. Purified plasmid DNA was digested with restriction enzymes EcoRI, BamHI or NcoI, according to the manufacturer’s protocol (Thermo Scientific, Waltham, MA, USA). The resulting DNA fragments were separated by electrophoresis in 0.8% agarose gels.

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Chmielowska C., Korsak D., Chapkauskaitse E., Decewicz P., Lasek R., Szuplewska M, & Bartosik D. (2021). Plasmidome of Listeria spp.—The repA-Family Business. International Journal of Molecular Sciences, 22(19), 10320.

Publication 2021

lysostaphin lysozyme EcoRI BamHI

Agarose Electrophoresis Gels Listeria Lysostaphin Lysozyme Plasmid Restriction enzymes ecori Rflp Strains

Corresponding Organization : University of Warsaw

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Variable analysis

independent variables

Isolation of plasmid DNA from Listeria spp. and B. subtilis strains
Use of lysostaphin instead of lysozyme for isolation of plasmid DNA from S. aureus strains

dependent variables

Sequence assembly of plasmid DNA verified by RFLP analysis
Digestion of purified plasmid DNA with restriction enzymes EcoRI, BamHI or NcoI
Separation of resulting DNA fragments by electrophoresis in 0.8% agarose gels

control variables

Isolation of plasmid DNA as described previously [23]
Large-scale preparation methods for plasmid DNA sequencing [80]
Manufacturer's protocol for restriction enzyme digestion (Thermo Scientific)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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