Plasmid DNA Purification and Contamination Screening

The plasmid DNA from both methods was pooled and subjected to overnight digestion at 37°C with exonuclease V Rec BCD (New England Biolabs, Ipswich, Massachusetts, United States) to remove chromosomal DNA. To test chromosomal DNA contamination PCR reactions using universal primers including the V4 region of 16S rRNA gene were performed. The primers used for bacteria were F5′-CCTACGGGNGGCWGCAG-3′ and R5′-GGATTAGATACCCBDGTAGTC-3′ (Klindworth et al., 2013 (link)); and for archaea F5′-CCCTAYGGGGYGCASCAG-3′ and R5′-ATTAGAKACCCSNGTAGTCC-3′ (Gantner et al., 2011 (link)). The plasmid DNA was purified using SureClean Plus kit as recommended by the manufacturer (BIOLINE, London, United Kingdom). Subsequently, concentration was measured by Qubit fluorometric quantification (Invitrogen, Thermo Fisher Scientific, CA, United States).