Fig. 2

Cloning of ASncmtRNA-2 and in vitro transcription. A Schematic diagram of ASncmtRNA-2. The 3 fragments that were joined together for cloning the complete sequence are shown in different colors: fragment 1 (red) was cloned between EcoRI and AflII sites and contained a T7 promoter sequence at its 5′ end (crosshatched red); fragment 2 (blue) was cloned between AflII and SacII sites; and fragment 3 (green) was cloned between SacII and HindIII sites (see also Additional file 1). The size of each fragment is shown in the numbers with the corresponding colors. Arrows and numbers represent the positions of the primers used for PCR (see Additional file 2). The correct and complete cloning of ASncmtRNA-2 was corroborated by EcoRI and HindIII digestion (B) and PCR (C). D The full-length plasmid (pAS2) was linearized and in vitro-transcribed (middle lane). Digestion of the transcript with RNase A yielded a band that migrates slightly above the 500 bp standard, which is consistent with the 551-bp dsRNA region. E The correct composition of the transcript was corroborated by RT-PCR. C, E Amplicon sizes are shown at the bottom of each gel