Kanamycin lb agar plates

The pMC O1P1-3C plasmid was produced by digestion of the pMC O1P1-HIV-3C^T plasmid with NotI-HF^® and EcoRI-HF^® (New England Biolabs) according to the manufacturer’s instructions. The nucleotide sequence encoding the FMDV 3C was derived by PCR amplification of a template plasmid containing the coding region of FMDV Asia Lebanon 1989 strain (GenBank accession no: JF739177). Ligation was performed using T4 DNA Ligase (Roche) and subsequently transformed into NEB^® 5-alpha competent E. coli (New England Biolabs) and plated on 50 µg/mL Kanamycin LB agar plates (Teknova). Selected colonies were grown in imMedia™ Growth Medium with Kanamycin (Invitrogen) overnight at 37 °C.

Previously constructed pTarget super-luminescent Gaussia princeps luciferase (SGLuc) plasmid [6 (link)] was used as a template for the insertion of SGLuc into pMC.CMV-MCS-SV40polyA with BamHI-HF^® and EcoRI-HF^®. Ligation was performed using T4 DNA Ligase (Roche), and subsequently transformed into NEB^® 5-alpha competent E. coli (New England Biolabs) and plated on 50 µg/mL Kanamycin LB agar plates (Teknova). Selected colonies were grown in imMedia™ Growth Medium with Kanamycin (Invitrogen) overnight at 37 °C.

Minicircle vector pMC.CMV-MCS-SV40polyA (System Biosciences, Palo Alto, CA, USA) and plasmid pUC57 O1P1-HIV-3C^T, synthesized by Genscript and containing the O1 Manisa P1 sequence, were digested with BamHI-HF^® and EcoRI-HF^® (New England Biolabs) as per the manufacturer’s instructions. Ligation was performed using T4 DNA Ligase (Roche) and subsequently transformed into NEB^® 5-alpha competent E. coli (New England Biolabs) and plated on 50 µg/mL Kanamycin LB agar plates (Teknova). Selected colonies were grown in imMedia™ Growth Medium with Kanamycin (Invitrogen) overnight at 37 °C.