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:
In the second step, the PCR products are added to the Gibson Assembly 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)
- In a PCR tube, mix the components on ice in the order they are listed above.
- Perform thermocycling program
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
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Relevant papers and books
- 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.