This is a specific protocol for creating a DNA construct that will unzip the "17-mer" sequence from plasmid pCP681 from Craig Peterson lab. Steve used this plasmid in his 2002 Biophysical Journal Paper (PMID 12124289). Originally designed to study nucleosome remodeling, this repetitive DNA is also convenient for studying site-specific DNA-binding proteins via unzipping, because multiple binding sites can be probed from a single-tether. The method here is easily adaptable to other DNA unzipping segments, if a few pitfalls are considered. The protocol below is a more detailed version of that provided in Koch et al. 2002.
The protocol is naturally sub-divided into three parts, followed by a ligation reaction(s).
Downstream unzipping segment
Ligation, final product formation
Optional End capping