Team:EPF-Lausanne/Tools/Gibson assembly

From 2011.igem.org

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We successfully used Gibson assembly for our plasmid assemblies, allowing us to put different genes in our plasmid backbones. You can find our protocol in our Notebook, under "Protocols" section.
We successfully used Gibson assembly for our plasmid assemblies, allowing us to put different genes in our plasmid backbones. You can find our protocol in our Notebook, under "Protocols" section.
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== Requirements ==
 
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In order to perform Gibson assembly on two DNA sequences, these sequences need to overlap by at least 20bp at the edge. You can easily add these overlaps by extension PCR
 
== Principle ==
== Principle ==
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Gibson assembly relies on the activity of 3 different enzymes:
Gibson assembly relies on the activity of 3 different enzymes:
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* Phusion polymerase
* Phusion polymerase
* Taq ligase
* Taq ligase
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In order to perform Gibson assembly on two DNA sequences, these sequences need to overlap by at least 20bp at the edge. You can easily add these overlaps by extension PCR.
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Combined together, these 3 enzymes allow overlapping DNA sequences (sequences A and B) to be ligated together in a single and isothermal reaction. First, the T5 exonuclease chews up bases at the 5' end on each strand, creating single-strands on the edges. Due to the sequence overlap, the single strand from A will anneal with the single strand from B. Then, the Phusion polymerase extend the DNA sequence from the 3' of the AB overlap. Finally, when there all the gaps have been filled by the polymerase, the Taq ligase ties up the A and B fragments.
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Combined together, these 3 enzymes allow overlapping DNA sequences (sequences A and B) to be ligated together in a single and isothermal reaction. First, the T5 exonuclease chews up bases at the 5' end on each strand, creating single-strands on the edges. Due to the sequence overlap, the single strand from A will anneal with the single strand from B. Then, the Phusion polymerase extends the DNA sequence from the 3' of the AB overlap. Finally, when there all the gaps have been filled by the polymerase, the Taq ligase ties up the A and B fragments.
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[[File:EPFL_Gibson_paper_picture.jpg]]
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[[File:EPFL_GibsonAssemblystrandscoloured.jpg|600px]]
== Step-by-step ==
== Step-by-step ==
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== Disadvantages ==
== Disadvantages ==
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* Primers are more complicated to design and longer - they are more expensive and take longer to synthesize
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* Primers are more complicated to design and longer - they are more expensive and take longer to be produced
* The reaction mastermix is expensive - due to large amounts of Taq ligase needed
* The reaction mastermix is expensive - due to large amounts of Taq ligase needed
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* A lot of negative resluts, du to the tendency of the backbones to close up themselves
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* A lot of negative results, due to the tendency of the plasmid backbone to close up on itself

Latest revision as of 21:41, 18 September 2011