Journal: PLoS ONE
Article Title: Experimental Pathways towards Developing a Rotavirus Reverse Genetics System: Synthetic Full Length Rotavirus ssRNAs Are Neither Infectious nor Translated in Permissive Cells
Figure Lengend Snippet: eGFP ssRNA species produced in vitro . ssRNAs were synthesised from PCR-derived amplicons with a T7 Pol promoter introduced at the 5′ end to facilitate in vitro transcription. PCR amplicons were digested with Bsm BI to define the 3′ end of the transcription cassette. Templates, either 500 ng or 1 µg, were incubated with T7 Pol in the absence or presence of a cap analogue using MEGAscript® or Mmessage Mmachine®. Uncapped ssRNAs were purified and post-transcriptionally capped using ScriptCap™ m7G Capping System. ssRNA was polyadenylated using E. coli polyadenylation polymerase (ePAP) Ambion. Lane R: RiboRuler™ High Range; lanes 1–6: approximately 250 ng each of eGFP ssRNA; uncapped, post-capped, uncapped polyadenylated, post-capped polyadenylated, co-capped, co-capped polyadenylated, respectively. 1.5% TBE AGE 60 V for –90 min. The polyadenylated RNA bands are less sharp as the molecules differ in the numbers of A residues added at the 3′end.
Article Snippet: For co-transcriptional capping of ssRNAs, the Mmessage Mmachine T7 kit (Ambion) was used with the following alterations: 1.5 µl of T7 RNA Pol (Ambion; 20 u/ul) per reaction was added, and the amount of linear template was increased to 1 µg for each reaction.
Techniques: Produced, In Vitro, Polymerase Chain Reaction, Derivative Assay, Incubation, Purification