Plasmid purification kit

The human VEGFA in a pcDNA3.1 + eGFP vector was purchased from GenScript. The EGFP-tagged VEGFA gene was excised from the original plasmid using EcoRI and XbaI Fast Digest restriction enzymes (Thermo Fisher, Waltham, MA, USA). The resultant genes of interest were run on a 1% (m/v) agarose gel containing SYBR Green (Thermo Fisher) at 100 Volts. After one hour, the DNA bands were visualized using a blue light transilluminator (miniPCR bio, Boston, MA, USA). Each gene fragment was excised and purified using NEB’s Gel Extraction Kit. The genes of interest were each ligated, using Instant Sticky End Ligase (NEB), into the pOET6 plasmid with compatible sticky ends. The VEGFA-eGFP-pOET6 plasmid was then chemically transformed into DH5alpha E. coli (Thermo Fisher) and selected for using the ampicillin resistance present within the pOET6 plasmid (MJS BioLynx Inc., Brockville, ON, Canada). Several different LB agar plates were streaked with different dilutions of the transformed bacteria and grown overnight. The next day, the resistant colonies containing the gene of interest were selected and grown overnight to amplify the plasmid. The plasmid was then extracted and purified using NEB’s plasmid purification kit. The resultant purified pOET6 plasmid, each with a gene of interest, was used for all future virus production steps or frozen at −20 °C for future use.