In vitro transcription and mRNA capping

The plasmid pET28a-MVHL-STOP2 was amplified by PCR using specific oligonucleotides (forward primer 5′-TCCGGCGTAGAGGATCGAGATC-3′, reverse primer 5′-GACTCGAGCAGATCTATTAAGAGCGGTCGGTAAAACTTCGGCCAGTGAATTTCAGTGGTATTTGTG-3′). The PCR product was transcribed in vitro using T7 RNA polymerase and purified by LiCl/EtOH precipitations. The purified mRNA was capped using Vaccinia Virus Capping Enzyme and the ScriptCap m7G Capping System (CellScript).