Team:Cambridge/Gibson/Protocol

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If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.
If you wish to ligate two pieces of DNA using Gibson they must be altered so as to have 40bp of overlap at the point of ligation.
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The standard way to do this is with PCR with specialised primers. We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]
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The standard way to do this is with PCR with specialised primers (see [[Team:Cambridge/Gibson/Mechanism |mechanism]]). We have designed a tool to help you do this: [http://www.gibthon.org Gibthon]
==Step 2: Order Primers==
==Step 2: Order Primers==
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==Step 3: PCR ==
==Step 3: PCR ==
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PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer:
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<div style="float:right; clear:both">&nbsp;</div>
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{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}
{{:Team:Cambridge/Templates/RightImage|image=Phusion.jpg|caption=Phusion Polymerase}}
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PCR is a bit of a dark art, but we have found that these general principles have served us well over the summer.
 
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The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.
The Annealing T<sub>m</sub> that should be used is the temperature of the main 20 or so bases of the primer (not including the flap), since the flap only begins to anneal after the first few cycles, by which point primer specificity is less of an issue.
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<i>e.g. If you were ligating two fragments (A and B) you could put:</i>
<i>e.g. If you were ligating two fragments (A and B) you could put:</i>
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Latest revision as of 20:31, 26 October 2010