Journal: Heliyon

Article Title: Maintenance and gene electrotransfer efficiency of antibiotic resistance gene-free plasmids encoding mouse, canine and human interleukin-12 orthologues

doi: 10.1016/j.heliyon.2022.e08879

Figure Lengend Snippet: Construction of the pORF-hIL-12-ORT plasmid. (a) Construction plan. (b) Annotated plasmid map confirmed by whole plasmid sequencing. c) Restriction analysis confirming the pORF-hIL-12-ORT plasmid construction: The plasmid was cut with Nhe I, Nco I and Hpa I restriction enzymes and its identity was confirmed by positive matching of the simulated pattern of bands created by SnapGene software (left) to the actual pattern on the electrophoretic gel (right). MW: molecular weight, GeneRuler DNA ladder Mix. Lane 1: Nhe I: 2596 bp, 1556 bp, 363 bp; lane 2: Nco I + Hpa I: 2648 bp, 1857 bp; lane 3: Kpn I: 2317 bp, 2198 bp.

Article Snippet: To visualize plasmid topologies, uncut plasmids were run (1 h, 100 V, PowerPac Basic Power Supply, BioRad) on un-stained 1% agarose gel together with supercoiled DNA Ladder (New England Biolabs).

Techniques: Plasmid Preparation, Sequencing, Software, Molecular Weight

Journal: Heliyon

Article Title: Maintenance and gene electrotransfer efficiency of antibiotic resistance gene-free plasmids encoding mouse, canine and human interleukin-12 orthologues

doi: 10.1016/j.heliyon.2022.e08879

Figure Lengend Snippet: Electrophoretic evaluation of plasmids. (a) Electrophoresis of 10 μL of minipep eluates from first to fifth passage linearized with Not I restriction enzyme. Plasmid lengths: pORF-mIL-12 (p40p35) - 4833 bp, pORF-mIL-12-ORT - 4380 bp, pORF-caIL-12-ORT - 4293 bp, pORF-hIL-12-ORT - 4515 bp. 1% agarose pre-stained with 1× SYBR tm Safe, 100 V, 1 h, LL, linear ladder: GeneRuler DNA ladder mix. Lanes 1–5, passages. (b) Electrophoresis of 200 ng of uncut plasmids isolated from first to fifth passage. 1% agarose, 100 V, 1 h, stained in 1× SYBR tm Gold. SCL, supercoiled ladder: Supercoiled DNA Ladder. SCm, supercoiled monomer. SCd, supercoiled dimer, SCt, supercoiled topoisomers. Lanes 1–5, passages (c) pORF-hIL-12-ORT plasmid incubated with increasing concentrations of SYBR tm Safe (0, 0.5×, 1× and 2×). SCL, supercoiled ladder: Supercoiled DNA Ladder.

Article Snippet: To visualize plasmid topologies, uncut plasmids were run (1 h, 100 V, PowerPac Basic Power Supply, BioRad) on un-stained 1% agarose gel together with supercoiled DNA Ladder (New England Biolabs).

Techniques: Electrophoresis, Plasmid Preparation, Staining, Isolation, Incubation