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. 2004 Oct 8;32(18):e140. doi: 10.1093/nar/gnh138

Figure 3.

Figure 3

Gel electrophoresis demonstrating the creation of dsRNA overhangs using the 5′ overhang protocol through the ligation to dsDNA fragments. In all gels, the left lane shows a DNA ladder with molecule lengths indicated in kb. (A) Result of the transcript reaction of a 4.2 kb dsRNA molecule with 5′ overhangs complementary to those of 0.4 kb dsDNA molecules: the more slowly migrating band only appears following transcription. (B) The transcription reaction following treatment with DNase I. The single remaining band must be dsRNA. (C) Ligation by T4 DNA ligase of the 4.2 kb dsRNA molecule with 5′ overhangs to 0.4 kb dsDNA ends tagged with biotin and digoxigenin. The dsDNA molecules were digested with BsmA1 to yield overhangs complementary to those incorporated into the 4.2 kb dsRNA molecule. The appearance of a new more slowly migrating band indicates the presence of molecules that have been ligated using the designed 5′ overhangs.