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Gibson Assembly

By René Davis, 2012


Contents

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:

Gibson Assembly step 1

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

Gibson Assembly step 2

Materials

Designing primers:

Gibson Assembly primers

For a 50 μL PCR reaction:

  • 35 μL H2O
  • 5 μL 10X PCR buffer
  • 5 μL 2mM dNTPs (each)
  • 1.5 μL 50mM MgCl2
  • 1 μL 50μM sense primer
  • 1 μL 50μM antisense primer
  • 1 μL 5nM DNA template
  • 0.5 μL TAQ DNA polyermerase

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. TH 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.


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