Team:Cambridge/Gibson/Mechanism

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(Difference between revisions)
(Creating overlapping DNA sequences)
(Creating overlapping DNA sequences)
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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.
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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We now perform two separate '''PCR'''s, 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.  
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.
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.

Revision as of 20:41, 27 October 2010