input template plasmid  (TaKaRa)

Journal: PLoS ONE

Article Title: Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

doi: 10.1371/journal.pone.0199563

Figure Lengend Snippet: A. The mutant constructs were first amplified with two partially overlapping primers designed to incorporate the desired alteration (shown in green), using the corresponding wild type block as a template. The input template DNA was then eliminated by digestion with the restriction enzyme DpnI. In the second step, the PCR-derived plasmid was circularized in an assembly reaction, which merged and sealed the overlapping ends. B. Oligonucleotide pairs that were used to generate the mutants are illustrated. The mutant block 1-derived constructs were E1B19K-null, E1B55K-null or modified to express an endogenous E1B55K protein with a c-terminal FLAG epitope. Similarly, the E4ORF3 open reading frame was disrupted by a mutation at the initiation codon or the introduction of a c-terminal FLAG tag. The targeted codons are boxed, single base mutations are shown in red. At positions where the initiation codon of the target protein overlapped with a codon for a different protein, base substitutions were selected so that the mutation would be silent with respect to the second open reading frame. C. hTERT-RPE1 cells were infected with the synthetic Ad5 virus and the E1B mutant viruses generated in this study. Cells were lysed 16 h post-infection, and assessed by immunoblot with antibodies against p53 and the FLAG epitope, as indicated. D. hTERT-RPE1 cells were uninfected (no virus) or infected with wild type Ad5 and the E4 mutants. For all viruses, the MOI was 100. GAPDH was probed as a loading control.

Article Snippet: Input plasmid DNA templates were digested with 20 U of the restriction enzyme DpnI (New England Biolabs) for 30 min at 37°C.

Techniques: Mutagenesis, Construct, Amplification, Blocking Assay, Derivative Assay, Plasmid Preparation, Modification, FLAG-tag, Infection, Generated, Western Blot