Koch Lab:Protocols/Unzipping constructs/17mer

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This is a specific protocol for creating a DNA construct that will unzip the "17-mer" sequence from plasmid pCP681 from [http://www.umassmed.edu/pmm/faculty/peterson.cfm Craig Peterson lab].  [[User:Steven J. Koch|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.
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The protocol is naturally sub-divided into three parts, followed by a ligation reaction(s).
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==Anchoring segment==
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==Adapter duplex==
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==Downstream unzipping segment==
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==Ligation, final product formation==
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==Optional End capping==
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==Tethering protocol==
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See:
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Revision as of 20:40, 14 June 2008


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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).

Contents

Anchoring segment

Adapter duplex

Downstream unzipping segment

Ligation, final product formation

Optional End capping

Tethering protocol

See:

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