Gel electrophoresis demonstrating the creation of dsRNA overhangs using the 5′ overhang protocol through the ligation to dsRNA fragments. In all gels, the left lane shows a DNA ladder with molecule lengths indicated in kb. ( A ) Left lane: results of a transcription reaction on two separate 4.2 kb PCR products: the more slowly migrating band only appears following transcription. Right lane: the same reaction following treatment with DNase I. The band corresponding to the PCR products has been eliminated by DNase digestion, leaving a 4.2 kb dsRNA molecule. ( B ) The 4.2 kb dsRNA molecule ligated by T4 DNA ligase to 0.4 kb dsRNA fragments, all with 5′ overhangs. Two additional, more slowly migrating bands have appeared, corresponding to ligation products. ( C ) Controls applied to distinguish hybridization from ligation using T4 DNA ligase. We carried out two separate reactions, one reaction in the absence of ligase, and another in the presence of ligase. Both cases result in a gel pattern similar to (B) prior to heating. However, following heating of the reaction in the absence of ligase (left lane), only the band corresponding to the 4.2 kb dsRNA molecule remained, whereas following heating of the reaction in the presence of ligase (right lane), both the 4.2 kb dsRNA band and a longer band remained. This demonstrated that ligase repaired the phosphodiester backbone. We estimate that approximately 10% of the 4.2 kb dsRNA molecules were ligated to at least one of the shorter fragments. ( D ) The same experiment as in (C), but using Rnl2. The reaction was incubated for 30 min, heated to 55°C (well above the T m ≈ 28°C for the 8 nt overhangs) for 15 min, rapidly chilled on ice, and run on gel. Left lane: in the absence of Rnl2; right lane: in the presence of Rnl2. Again, the fact that longer bands remain in the right lane following heating indicates the presence of ligation products. The use of Rnl2 appears to increase the ligation efficiency to ≥50%.