Team:Cambridge/Gibson/Mechanism

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===Creating overlapping DNA sequences===
===Creating overlapping DNA sequences===
Overlapping DNA sequences can be created by PCR.  We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.
Overlapping DNA sequences can be created by PCR.  We can add twenty base-pairs to the end of a sequence by using a primer which runs as follows from 5' to 3'.
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<pre>
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20 bp of sequence to add -> 20 bp of template to anneal to.
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[[Image:PCR.png|500px|center|template extension using primers]]
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</pre>
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By using two such primers we can add 20 bp of sequence A to sequence B and 20 bp of sequence B to sequence A.  We are then ready to use Gibson Assembly. The Cambridge team have developed [http://www.gibthon.org/ Gibthon] to help you design primers for Gibson Assembly.  The tool allows you to put in two sequences and choose 20bp of each to get a 40bp primer; it then analyses the melting temperature and secondary structure of this primer.
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If we wish to ligate two sequences called '''A''' and '''B''' (in that order) we need to ensure that there is an overlap of 40bp: i.e. the end of sequence A has the same 40 bp as the beginning of sequence B.
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We now perform two separate PCRs, one on A and one on B. The first adds the beginning 20 bp of B to sequence A by using a '''~40nt primer''' as detailed in the figure above (Note: the diagram shows both ends being extended -often necessary to perform multiple ligations at once). The second adds the final 20 bp of A to sequence B. The end of A and the beginning of B now are composed of the '''same 40bp sequence''' (end of A + beginning of B), and we are now ready to use Gibson Assembly.  
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The Cambridge team have developed [http://www.gibthon.org/ Gibthon] to help you design primers for Gibson Assembly.  The tool allows you to put in two sequences and choose 20bp of each to get a 40bp primer; it then analyses the melting temperature and secondary structure of this primer.
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<html><div style="text-align:center"><img src="https://static.igem.org/mediawiki/2010/a/a3/Cambridge-Gib1.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html>

Latest revision as of 20:41, 27 October 2010