Haynes:Gibson Assembly

Gibson Assembly

Overview

The Gibson Assembly method allows you to assemble multiple DNA parts in just 2 steps.

Materials

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. 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

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References

Relevant papers and books

1. Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
2. Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
3. Ptashne, M (2004) Genetic Switch: Phage Lambda Revisited - Cold Spring Harbor Laboratory Press ISBN 0879697164

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