Haynes:Gibson Assembly

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Gibson Assembly

by René Davis, 2012


The Gibson Assembly method allows you to assemble multiple DNA parts in just 2 steps.
In the first step, 20 basepair overlaps are added to the sequences to be assembled. In the example below, I am assembling two proteins joined by a 60 basepair linker:

Gibson Assembly step 1

In the second step, the PCR products are added to the Gibson Assembly Mix:

Gibson Assembly step 2


Designing primers:

Gibson Assembly primers

PCR mix
15ul of Gibson Assembly Mix (I will add the recipe soon, but for now, find 15ul aliquotes in PCR tubes in a clearly labeled bag in the -20C freezer door... soonish)


  1. In a PCR tube, mix the components on ice in the order they are listed above.
  2. Perform thermocycling program

For now: http://django.gibthon.org/help/gibson/


Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

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Relevant papers and books

  1. Gibson, DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO (2009) - Nature Methods 6(5) 343-5 PMID 19363495


  • René at rene.davis at asu dot edu

or instead, discuss this protocol.