Team:Cambridge/Gibson/Mechanism

From 2010.igem.org

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(Creating overlapping DNA sequences)
(Creating overlapping DNA sequences)
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[[Image:PCR.png|500px|center|template extension using primers]]
[[Image:PCR.png|500px|center|template extension using primers]]
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By using two ~40nt oligonucleotides as 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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By using two ~40nt oligonucleotides as primers, we can add the final 20 bp of sequence A to sequence B and the beginning 20 bp of sequence B to sequence A.  We are then ready to use Gibson Assembly.  
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<html><div style="text-align:center"><img src="http://2010.igem.org/wiki/images/a/a3/Cambridge-Gib1.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html>
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Note:
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The end of sequence A with the extension now looks like:
The end of sequence A with the extension now looks like:
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This results in there now being a total overlap of 40bp between the two fragments, enough to perform Gibson assembly.
This results in there now being a total overlap of 40bp between the two fragments, enough to perform 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="http://2010.igem.org/wiki/images/a/a3/Cambridge-Gib1.png" style="border:1px solid gray; margin-top:20px; margin-bottom:20px;"></div></html>
== Gibson Assembly==
== Gibson Assembly==

Revision as of 20:30, 27 October 2010