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Gibson Assembly

By René Davis, 2012

Overview

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

Materials

Designing 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)

Procedure

1. In a PCR tube, mix the components on ice in the order they are listed above.
2. Perform thermocycling program
   1. 95 °C 5 min
   2. 95 °C 30 s
   3. T_H 30 s
   4. 72 °C 1 min for each 1 kb PCR product
   5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
   6. 72 °C 5 min
   7. 12 °C hold

Notes

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.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

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

Contact

• René at rene.davis at asu dot edu

or instead, discuss this protocol.