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.
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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==Individual segments==
The protocol is naturally sub-divided into three parts, followed by a ligation reaction(s).
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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===Anchoring segment===
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==Adapter duplex==
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===Adapter duplex===
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==Downstream unzipping segment==
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===Downstream unzipping segment===
==Ligation, final product formation==
==Ligation, final product formation==

Revision as of 19:43, 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.

Contents

Individual segments

The protocol is naturally sub-divided into three parts, followed by a ligation reaction(s).

Anchoring segment

Adapter duplex

Downstream unzipping segment

Ligation, final product formation

Optional End capping

Tethering protocol

See this example protocol.

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