T7 polynucleotide kinase

DNA sequences for making the DNA constructs used in this study can be found in Supplementary Table 2. DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). J7, unlabeled J7 and J7E were annealed by mixing the four strands with the molar ratio 1:1:1:1 (final concentration ~10 μM each) in 10 mM Tris:HCl (pH 8.0) and 50 mM NaCl followed by slow cooling from 90°C to room temperature for ~ 2 hours. J3 was prepared by mixing equimolar amounts of the four component oligonucleotides in PNK buffer (New England Biolabs), labelling them with γ-³²P-dATP (Perkin Elmer) and T7 polynucleotide kinase (New England Biolabs) followed by slow cooling from 90°C to room temperature for ~ 2 hours. 22-bp dsDNA was annealed by mixing the two strands with the molar ratio 1:1 (final concentration ~10 μM each) in 10 mM Tris:HCl (pH 8.0) and 50 mM NaCl followed by slow cooling from 90°C to room temperature for ~ 2 hours. J5m, n-J5m and J0m were constructed as previously described ^{7 (link),37 (link),45} . When J5m undergoes spontaneous branch migration, there are six possible donor-acceptor separations of 10, 12, 14, 16, 18, 20 bp for different branch positions. The donor-acceptor separation for J0m is 16 bp.