Circularization by T4 Rnl1 of the dsRNA obtained by pfRCT. (A) Schematic diagram of the ligation strategy. (B) Circularization results analyzed by dPAGE (8%, 8 M urea). Conditions for pfRCT: [Template] = 50 nM, [NTP]each = 2 mM, [T7 RNAP] = 2 U/µL, [RNase Inhibitor] = 2 U/µL, 1× T7 RNAP buffer, [RNase H] = 0.25 U/µL, [Helper] = 1 µM, 37°C for 4 h. Lanes 1 and 5: dsDNA templates; lanes 2 and 6: dsRNAs produced using pfRCT; lanes 3 and 7: the products in lanes 2 and 6 were ligated by T4 Rnl1; lanes 4 and 8: ligation products in lanes 3 and 7 were further digested using RNase R. Conditions for ligation: 50% (volume) of transcription solution, [T4 Rnl1] = 0.5 U/µL, 0.5× T4 Rnl1 buffer, [ATP] = 1 mM, [RNase Inhibitor] = 2 U/µL, 37°C, 6 h.
