miniprep column  (Qiagen)

Journal: bioRxiv

Article Title: Outpacing E. coli : Development of Vibrio natriegens as a Next-Generation Cloning Host

doi: 10.64898/2026.02.11.705132

Figure Lengend Snippet: The commonly used pUC19 plasmid and a 7.4kb lentiviral transfer plasmid containing long terminal repeats (LTRs) were transformed into either a strain lacking only dns ( recA + ), or a strain lacking both dns and recA ( recA - ). Two independent colonies of each strain/plasmid combination were cultured in liquid medium followed by plasmid isolation via miniprep. Purified DNA samples were analyzed by agarose gel electrophoresis. The monomeric, supercoiled plasmid species is indicated as “Desired plasmid”, while higher-molecular-weight, concatenated species are indicated as “Undesired multimers”. In the case of both plasmids, the recA deletion leads to a marked reduction in plasmid concatenation.

Article Snippet: Cells were processed through a Qiagen QIAprep Spin Miniprep column following the manufacturer instructions with the following modifications: the optional PB wash step was used, and plasmid was eluted by 2 x 50 μL elutions with TE buffer to ensure complete recovery of plasmid from the column matrix.

Techniques: Plasmid Preparation, Transformation Assay, Cell Culture, Isolation, Purification, Agarose Gel Electrophoresis, Molecular Weight

Journal: bioRxiv

Article Title: Outpacing E. coli : Development of Vibrio natriegens as a Next-Generation Cloning Host

doi: 10.64898/2026.02.11.705132

Figure Lengend Snippet: Colonies of strains on agar plates with and without the dns::oxyR2 modification carrying the plasmid pcDNA3.1 were inoculated into 2 mL aliquots of media in 24-well deep-well plates. Cultures were grown at 37°C with agitation at 300 RPM and humidity at 80% for 6 hours. Plasmid DNA was extracted from cell pellets from 100 μL of culture using Qiagen Miniprep columns, and plasmid DNA was quantified using a Qubit fluorometer. As seen from the figure, the presence of the dns::oxyR2 genetic modification increased plasmid yield at the 6 hr timepoint.

Article Snippet: Cells were processed through a Qiagen QIAprep Spin Miniprep column following the manufacturer instructions with the following modifications: the optional PB wash step was used, and plasmid was eluted by 2 x 50 μL elutions with TE buffer to ensure complete recovery of plasmid from the column matrix.

Techniques: Modification, Plasmid Preparation

Journal: bioRxiv

Article Title: Outpacing E. coli : Development of Vibrio natriegens as a Next-Generation Cloning Host

doi: 10.64898/2026.02.11.705132

Figure Lengend Snippet: All strains were transformed with pcDNA3.1 via chemical transformation and plated on agar plates (NBx CyClone™ agar for NBx CyClone™, and LB for E. coli ). Agar plates were incubated overnight, followed by colony picking into 2 mL aliquots of liquid media with carbenicillin in wells of a 24-well deep-well plate (In this preliminary experiment, DOE condition 14 (see Supplementary Text 8) media was used for NBx CyClone™, LB for E. coli ). The plate was incubated at 37°C for 6 hours with agitation at 300 RPM in a shaker with a 25 mm orbit. (A) Growth curves measured directly in the 24-well plate via an ESSItech mtpOD online biomass monitor. (B) Average volumetric yields (in mg/L) of pcDNA3.1 after 6 hours of cultivation isolated by Qiagen miniprep columns and measured via fluorometry (Qubit). Averages were calculated from yields from four independent colonies (n=4) from each strain. (C) Plasmid quality assessed by a supercoiled agarose gel stained with SYBRsafe stain. The supercoiled gel allows visualization of other plasmid topologies such as undesired concatenated multimers.

Article Snippet: Cells were processed through a Qiagen QIAprep Spin Miniprep column following the manufacturer instructions with the following modifications: the optional PB wash step was used, and plasmid was eluted by 2 x 50 μL elutions with TE buffer to ensure complete recovery of plasmid from the column matrix.

Techniques: Transformation Assay, Incubation, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

Journal: bioRxiv

Article Title: Outpacing E. coli : Development of Vibrio natriegens as a Next-Generation Cloning Host

doi: 10.64898/2026.02.11.705132

Figure Lengend Snippet: Colonies of NBx CyClone™ strains harboring various plasmids were inoculated into 2 mL of media (NBx CyClone™ growth media) in 24-well deep-well plates and cultivated in a shaking incubator at 250 – 300 RPM at 37°C for 6 hours. Volumetric yields determined by harvesting 200 μL aliquots of each culture and processing through a Qiagen QIAprep® miniprep column. Plasmid concentrations were measured using a Qubit fluorometer.

Article Snippet: Cells were processed through a Qiagen QIAprep Spin Miniprep column following the manufacturer instructions with the following modifications: the optional PB wash step was used, and plasmid was eluted by 2 x 50 μL elutions with TE buffer to ensure complete recovery of plasmid from the column matrix.

Techniques: Plasmid Preparation

Journal: bioRxiv

Article Title: Outpacing E. coli : Development of Vibrio natriegens as a Next-Generation Cloning Host

doi: 10.64898/2026.02.11.705132

Figure Lengend Snippet: Individual colonies of NBx CyClone™ transformed with pUC19 were inoculated into different growth formats (25 mL culture volume in 125 mL baffled flask; 4 mL or 2 mL in a 24-well deep-well plate; and 1 mL or 0.5 mL in a 96-well deep-well plate). Each condition was tested in triplicate. All cultures were incubated at 37°C for 4, 6, or 8 hours. The flasks were agitated at 250 RPM with a 2.5 cm orbit, the 24-well deep-well plate at 300 RPM with a 2.5 cm orbit, and the 96-well deep-well plate at 1000 RPM with a 3 mm orbit. (A) Culture density (OD600) of each culture at different timepoints. (B) Volumetric yields (mg/L) determined by harvesting 200 μL aliquots of each culture and processing using a Qiagen QIAprep® miniprep kit. Plasmid concentrations were measured using a Qubit fluorometer. (C) Total plasmid (in µg) recovered from the respective culture volumes at 4, 6, and 8 hours.

Article Snippet: Cells were processed through a Qiagen QIAprep Spin Miniprep column following the manufacturer instructions with the following modifications: the optional PB wash step was used, and plasmid was eluted by 2 x 50 μL elutions with TE buffer to ensure complete recovery of plasmid from the column matrix.

Techniques: Transformation Assay, Incubation, Plasmid Preparation