Plasmid plus maxi kit

Expression minigene plasmids were created as previously described [32 (link)]. CFTR-EMGi1-i5 contained abridged intron 1 (216 bp of 5′ and 212 bp of 3′), abridged intron 2 (311 bp of 5′ and 264 bp of 3′), abridged intron 3 (374 bp of 5′ and 456 bp of 3′), abridged intron 4 (307 bp of 5′ and 333 bp of 3′), and full-length intron 5 (882 bp). E60X, L88X, and Y122X variants were individually introduced to EMG plasmids through site-directed mutagenesis (SDM) as previously described [32 (link)]. Mutagenic primers were designed using QuickChange Primer Design tool. Primers were used to PCR-amplify EMG plasmid, followed by digest with DpnI, transformation of XL10-Gold ultracompetent cells (Agilent, Cedar Creek, TX, USA), and selection of colonies on LB-Ampicillin plates (Quality Biologicals, Gaithersburg, MD, USA). DNA minipreps were prepared (Denville Spinsmart Plasmid Miniprep DNA Purification Kit, Swedesboro, NJ, USA) and presence of the variant of interest was confirmed by Sanger sequencing. Selected miniprepped plasmid was used to transform XL10-Gold ultracompetent cells, and DNA maxipreps were prepared (Qiagen Plasmid Plus Maxi Kit, Hilden, Germany). Sanger sequencing was used to verify sequence of entire plasmid and confirm presence of the variant of interest.

For the construction of the NiV DNA vaccine (also termed DNA-G), The optimized gene was subcloned into the clinically used vector pVAX1 between the restriction sites KpnI and BamHI with the Kozak sequence incorporated at the 5’ end of the genes. The plasmid was transformed into E. Coli Top 10 cells for plasmid amplification and purified using an endotoxin-free Plasmid Plus Maxi Kit (Qiagen).

For routine growth of plasmids, we used chemically competent Stable E. coli cells (#C3040I; NEB, Ipswich, MA, USA). DNA for transfection was prepared using the endotoxin-free grade Plasmid Plus Maxi Kit (#12963; Qiagen, Hilden, Germany) or ZymoPURE Plasmid Midiprep Kits (#11-550; Zymo Research, Irvine, CA, USA). The purified plasmid was resolved by agarose gel electrophoresis to rule out RNA or genomic DNA contamination. Plasmids used for quantitative studies of HDR efficiency were never thawed more than 3 times to prevent unwanted degradation of plasmids.